Protein hydrolysates from fish proteins are sources of peptides that may present bioactive properties such as antioxidant, antihypertensive, antimicrobial, or antidiabetic. These bioactivities can be beneficial to both human health and the food industry. For this reason, a study to obtain bioactive peptides from a by-product from fish processing becomes interesting as a useful method to add value and to minimize the environmental impacts caused by the mismanagement and inappropriate disposal of these by-products. The fish mechanically separated meat (MSM) is a by-product obtained from the process of separating the muscle from bones, consisting in portions of free-viscera muscle, bones and skin. In this context, the Blackfin pacu (Colossoma macropomum) stands out for the great commercialization of cuts such as band and boneless fillets, which generates by-products from which can be obtained the MSM. The objective of this study was to evaluate the antioxidant activity presented by the protein hydrolysate obtained from Blackfin pacu MSM using the enzyme Alcalase. For the evaluation of the antioxidant activity, two concentrations of protein hydrolysate (5 and 10 mg/mL) and the following analytical methods were used: DPPH radical scavenging activity (%), reducing power (absorbance) and hydroxyl radical scavenging activity (%). The results were statistically evaluated through analysis of variance (ANOVA) and the means compared by the Tukey test at 5% significance. The antioxidant activity showed a significant dose-dependent relationship (p <0.05) for all the methods evaluated (DPPH: 19.6% and 22.3%; Reducing power: 0.226 and 0.444; Hydroxyl radical scavenging: 94.3 and 95.7%, for 5 and 10 mg/mL respectively). Based on these results, it can be concluded that Blackfin pacu MSM protein hydrolysate possess a great potential as a source of antioxidant compounds, which could be used as nutraceuticals and/or ingredients for functional foods.