Background: Drop jump (DJ) is a kind of plyometric training. There is little research on the effect of drop height (DH) on unilateral DJ. This study explored the optimal DH of DJ by unilateral lower limb, aiming to provide a reference for males with different exercise levels to avoid the imbalance between bilateral lower limbs and for coaches to develop accurate and effective unilateral DJ training programs. Methods: 18 college athletes and 13 physically active students were recruited to participate in this study and then asked to take three tests, namely “single-leg DJ at low height (L- SLDJ)”, “single-leg DJ at medium height (M-SLDJ)”, and “single-leg DJ at high height (H-SLDJ)”. Motion Capture System and Force Plate were used to synchronously collect motion parameters. Results: At M-SLDJ, both dominant and non-dominant legs performed significantly better than those at the other two types of DH in terms of leg stiffness and power for males with different exercise levels. At H-SLDJ, for males with different exercise levels, dominant leg had significantly better performance in leg stiffness, power etc. than non-dominant leg. Conclusions: The M-SLDJ designed in this study can be used by college athletes or students majoring in physical education for unilateral lower limb DJ training to maximize the performance of dominant or non-dominant leg. Due to the significant difference between two legs in the case of H-SLDJ, the eccentric contraction ability and stability of the non-dominant leg should be enhanced during the training to avoid bilateral imbalance.