This present work aimed to evaluate the effect of threonine fermentation biomass (TFB) as an alternative protein source to replace the fishmeal (FM) in diets for Litopenaeus vannamei on shrimp growth and apparent digestibility. Four fish oil-free diets were formulated to contain 350 g kg−1 of crude protein with different TFB levels in replacement of FM (g kg−1): (1) 0, control; (2) 35.9, low (L-TFB); (3) 71.8, medium (M-TFB); (4) 107.7, high (H-TBF). A 42-day feeding experiment was evaluated in triplicate with 60 juveniles (4.94 ± 0.34 g) distributed aleatory into 12 20-L tanks and fed four times a day at 8% of the total biomass. Faeces were collected daily by siphon four times a day after the first week of the feeding experiment to determine the apparent dry matter and protein digestibility coefficients (ADMD and APD respectively) using acid insoluble ash as an internal marker. Growth performance was not affected, but higher ADMD and APD were recorded in the M-TFB. Thus, FM was replaced up to 12% with TFB, achieving the formulation of a fish-free diet with a proper balance between bacterial, animal and plant ingredients without compromising its digestibility and shrimp growth performance.