Understanding the impact of gene action and combining ability on targeted traits in a hybrid breeding program is imperative. The objective of this study was to estimate the genetic effect and combining ability of S2 super sweet corn lines on carotenoid content. Twenty S2 lines were crossed with two hybrid testers by using the line × tester mating design. Regarding parents, forty hybrids and two commercial checks were evaluated for carotenoid content across two different growing seasons between 2019 and 2020. The result indicated that the non-additive gene action governed the inheritance of carotenoid content. Several promising S2 lines for individual carotenoids were identified; only L20 possessed different and positive GCA values for all observed carotenoids. Moreover, genotype T2 was a promising tester to identify superior lines for creating biofortified sweet corn hybrids. Testcross hybrids with satisfactory performance, desirable SCA estimates, and involving at least one of the pairwise parents with positive and high GCA were successfully defined. Hybrid T2 × L20 had high lutein, zeaxanthin, β-cryptoxanthin, and total carotenoids contents (ranging from 12.58 to 74.01 µg/g of dry weight), whereas hybrid T2 × L9 showed the highest content of β-carotene (4.19 µg/g of dry weight). We propose that high GCA and line at least one of the pairwise parents be included in indirect selections for the hybrid breeding of high-carotenoid sweet corn.