Forbes said Vergara's role as Gloria in Modern Family and some lucrative product endorsements helped her earn $43m (£32.6m) in the last 12 months.
It marks the fifth year the Colombian-American actress has topped the chart.
Forbes also said she earned more than any of her male counterparts in the past year.
The Big Bang Theory's Kaley Cuoco was the second-highest paid actress, earning $24.5m (£18.6m).
Cuoco tied with Vergara at the top of last year's Forbes list, when both actresses earned $28.5m (£21.6m).
The Mindy Project's Mindy Kaling is the biggest climber in this year's chart. Her earnings of $15m (£11.4m) helped her to rise from eighth place in 2015 to third this year.
Mariska Hargitay, who appears in Law & Order: Special Victims Unit, and Grey's Anatomy star Ellen Pompeo rounded off the top five.
Source: Forbes
This year's highest new entry on the Forbes list was Priyanka Chopra, who appears in ABC drama Quantico. She was the eighth highest earner with $11m (£8.4m).
Chopra, who is well known in India, is set to become more familiar to western audiences next year when she stars in Baywatch alongside Dwayne Johnson - the world's highest paid actor.
Scandal star Kerry Washington, Stana Katic from Castle, The Good Wife's Julianna Margulies and Vergara's Modern Family co-star Julie Bowen also featured in this year's top 10.
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Modern Family star Sofia Vergara has retained her title as the highest paid actress on US television, according to the latest Forbes magazine rich list.