Ronaldo got away from his marker with some quick footwork before rolling the ball across for Mario Mandzukic to tap into the net in the 59th minute.
The 33-year-old Ronaldo has been involved in 44 percent of Juve's goals this season, having scored 10 and assisted six others. And Mandzukic is clearly reaping the benefits. The Croatia international scored five goals in 32 league matches last season and already has six in 10 this campaign after developing an impressive understanding with Ronaldo.
"Mandzukic is scoring because maybe with Dybala he played in one way, where Dybala was benefiting from his movements, and now he's benefiting from the way Ronaldo plays," Juventus coach Massimiliano Allegri said.
Valencia put in a spirited performance in Turin but it came to nothing as Marouane Fellaini scored in stoppage time to give Manchester United a 1-0 win over Young Boys and secure its position in the round of 16.
Fellaini touched the ball with his hand just before scoring and Valencia coach Marcelino said the implementation of the video review system in the Champions League can't come fast enough.
"They told me there was handball, but there's nothing we can do about it," Marcelino said. "But that's football. It's made up of details. What is strange is that we don't yet have VAR in such a big competition as this."
VAR is already in place in Italy and Spain and will be used in the Champions League from next season but its introduction could be brought forward to this campaign's knockout stages. UEFA's executive committee could make a fresh decision at a Dec. 2-3 meeting in Dublin.
Juventus has a two-point advantage over United and will secure top spot in Group H with a win over Young Boys on Dec. 12. If it draws, a United win would see the English team top the group thanks to having scored more away goals in the matches between the two teams.
Juventus only needed a point against Valencia to book its spot in the last 16 but the Bianconeri were keen to get back on track following a surprise home loss against Manchester United in the last round of group matches.
Ronaldo had scored his first Champions League goal for Juventus in that game and looked eager for more against Valencia, having been sent off against the Spanish team when the two sides met in September — in a match Juventus went on to win 2-0 despite its numerical disadvantage.
The Portugal star had two chances in the opening three minutes but sent one effort wide of the left post and the other straight at former Juventus goalkeeper Neto.
Ronaldo had another good chance midway through the first half following good interlinking play between Paulo Dybala and Alex Sandro but again fired wide.
Valencia was mainly camped out in its own half, defending in numbers, but had the best chance of the first half when Mouctar Diakhaby headed a corner toward goal only for Wojciech Szczesny to pull off a stunning save at point-blank range.
Juventus finally broke the deadlock in the 59th minute. Valencia defender Gabriel knew precisely what Ronaldo was about to do but was powerless to prevent him and let out a roar of frustration after the goal.
Valencia thought it had equalized immediately but Diakhaby used his hand to punch the ball into the back of the net and was instead shown a yellow card.
It was another record for Ronaldo in the Champions League as he became the first player ever to reach 100 wins in the competition.
Kathryn Mayorga filed a civil lawsuit in September in Nevada claiming Ronaldo raped her in his Las Vegas hotel room in 2009. Police reopened an investigation into the allegation at her request. Ronaldo has denied any wrongdoing.