Tops-Alexander, who had a baby girl in July, and her feisty mare California won a five-horse jump-off with a clear round in 32.07 seconds. Bassem Mohammed of Qatar finished in second place, followed by Ireland's Darragh Kenny and Cassini Z.
"It's amazing, I wasn't here last year because I was pregnant, so nice to be back again," Tops-Alexander, a two-time LGCT overall winner, said in a televised interview. "I really didn't think I'd won actually, I had to keep looking at the screen."
As she entered the compact arena, built just meters from the ocean, as the last rider of the jump-off, it was, in many ways, as if the Australian had never been away.
Under pressure, Tops-Alexander delivered, producing a fast-paced yet skillful clear round, and finishing more than three seconds ahead of the then leader, Qatar's Mohammed.
After competing in the huge Campo Marte grass arena in the heart of Mexico City at high altitude two weeks ago in the first stop on the Global Champions circuit, California had not been feeling well, but a visit to the chiropractor had worked wonders for the mare.
Tops-Alexander, who is married to former Dutch Olympic champion and Global Champions founder Jan Tops, was full of praise for her horse and the course builder.
"All weekend, it's been not massive, very delicate, tricky, it's caught out a lot of riders in all different places," she said. "It was just a matter of getting your skills and having your horse in gear and ready, and today my horse, she was ready. I'm very fortunate to have her."
Tops-Alexander, who also won in Miami Beach in 2016, is trying to become the first rider to win the LGCT three times. Her win in Miami Beach means she moves up to third place in the overall standings with 54 points. Britain's Scott Brash, the winner in Mexico City, is in the lead with 67 points, followed by Belgium's Pieter Devos with 60 points.
Prague Lions win team event
Earlier on Saturday, the Prague Lions, with Czech rider Anna Kellnerova aboard Classic and Holland's Gerco Schroder with Glock's London, won the Global Champions League team competition with a total of eight penalties, ahead of Valkenswaard United in second place with 10 penalties and Monaco Aces in third with 14 penalties.
The Monaco Aces had taken the lead overnight after a double clear, but dropped two spots when French rider Julien Epaillard and his horse, Safari D'Auge, produced 12 penalties in the second round.
Valkenswaard United now lead the overall standings after two events.
All 19 teams are trying to qualify for the inaugural GC Play Offs in Prague just before Christmas.
The Czech capital will also stage the first Longines Global Champions Super Grand Prix, which will see the winners of each Tour leg facing off against each other.
After its first two events in Mexico and the US, the tour will move to Asia for the Shanghai leg between April 20 and 22.