Hanyu is bidding for a fifth national title in Tokyo with Uno hoping to make it four in a row having won the last three in Hanyu's absence.
Both skated in the penultimate group with Uno producing his best short program since taking silver at PyeongChang 2018 to go into the lead.
But double Olympic champion Hanyu had a clean skate, unlike at the Grand Prix Final in Turin earlier this month, to take victory with 110.72 - better than his world record of 110.53 set at last year's Rostelecom Cup.
As this is a national event, these scores are not recognised by the ISU for record purposes.
Junior Grand Prix Final winner Shun Sato showed why he is very much a star of the future with third place.
In his last singles competition before switching to ice dance, five-time Japanese champion and Vancouver 2010 bronze medallist Daisuke Takahashi ended the day in 14th position.
Uno back on track
That partnership already appears to be paying dividends with Uno, who turned 22 on Tuesday, smiling again during what has been a difficult season.
After parting company with long-time coaches Machiko Yamada and Mihoko Higuchi, and visiting Eteri Tutberidze's summer training camp, Uno finished eighth in the Internationaux de France before taking fourth at the Rostelecom Cup.
And he was back to his best here, starting with a sublime quad flip before landing a quad toe loop-double toe loop combination and then a triple Axel to score 105.71 and put him on track for a place at March's World Championships in Montreal.
Hanyu skates clean to take short program
With coaches Brian Orser and Ghislain Briand back on the boards after Briand's passport issues saw him miss the short program in Turin, Hanyu had made a change to his routine.
He swapped his last two elements in the program with his quad toe loop-triple toe loop combination now preceding the triple Axel.
While that means a score reduction of 0.57 points, today's superior execution made that something of an irrelevance.
The 25-year-old just about held on to his closing triple Axel in a superb skate to 'Otonal' which gives him a lead of five points over nearest rival Uno.
Sato confirms Turin impression
Shun Sato sprung something of a surprise at the Junior Grand Prix Final in Turin, setting a new world junior record in the free skate to take an emphatic win.
Before that competition, Yuma Kagiyama had beaten Sato by over 36 points in the junior nationals and he will represent Japan at the Lausanne 2020 Winter Youth Olympic Games.
Kagiyama disappointed again here in Tokyo, getting his planned opening triple Axel all wrong as he scored 77.41 to finish the day down in seventh place.
But Sato built on the impression he made at the JGP Final, landing a quad toe loop - not permitted in junior competitions - on his way to a score of 82.68 and third place.
Keiji Tanaka is fourth with 80.90 ahead of Koshiro Shimada on 80.59.
The free skate takes place on Sunday.