To establish an in vitro regeneration system for Cyperus esculentus L., its apical buds were used as explants, and MS was selected as the basic medium. An orthogonal experimental design was adopted to study the effects of 6-BA, TDZ, and ZT combinations on the proliferation of cluster buds of Cyperus esculentus L. The results showed that the order of influence of the three plant growth regulators on the proliferation of cluster buds was 6- BA > TDZ > ZT. The optimal medium for cluster bud induction was MS + 0.5 mg/L 6-BA + 0.5 mg/L TDZ + 0.7 mg/L ZT. The optimal medium for adventitious root induction was hormone-free MS medium, with a rooting rate of 100% and an average of 5.7 roots per explant. This study provides important technical support for the efficient cultivation of high-quality seedlings and the optimization of germplasm resources of Cyperus esculentus L.