The former One Direction star had been due to perform on the programme, before pulling out after the shock death of his 18-year-old sister earlier this week.
During the live show, Comic Relief founder Lenny said: “Louis Tomlinson wanted to be on this stage to help us but the sudden and tragic loss of his sister Felicite obviously means he can’t be here.
“The love and best wishes of all of us at Comic Relief go out to Louis and his family at this terrible time.”
Lenny also offered his thoughts to the families of those killed in the Christchurch mosque attacks, stating: “Can I just say at this moment how profoundly we share everyone’s sorrow about recent events in New Zealand.
“We send our condolences and respects to the families, and stand shoulder to shoulder, despite those who wish to do harm.”
This year’s Red Nose Day raised over £63 million for charity and the funds will go towards supporting projects both here in the UK and abroad.
The evening’s sketches and performances were punctuated by appeal films and reminders of what the night is really about, with one segment seeing Lenny call on viewers to help foodbanks providing for families in the UK.
“It’s hitting a huge number of people in this country right now,” he said. “Parents who can’t feed their children without help from charities and foodbanks.
“I’ve been talking about global poverty since Comic Relief began, but if you told me 10 years ago that I would be here today, asking you to reach into your pocket so we can help feed children in the UK, one of the richest countries in the world, I wouldn’t have believed you.
“But that is where we are, and that is exactly what I’m doing.”