Foods that contain natural antioxidants can improve people's health and performance and make them more resistant to environmental stressors like diseases, lousy living conditions, and heat stress. Marine algae are well known for their function as an antioxidant. Colaconema formosanum is a new rhodophyte discovered in Taiwan and its bioactive activities are rarely studied. This work aims to prepare the protein hydrolysate fraction with antioxidant properties in C. formosanum using single enzyme or complex enzyme hydrolysis. Afterward, a 3 kDa molecular weight cut-off (MWCO) was used for centrifugation, and then fractionation was carried out using Strong Cation Exchange (SCX) Chromatography. The quantification of antioxidant activity was performed using the DPPH test. The results showed that the thermolysin hydrolysate (IC50 1.48 ± 0.92 mg/mL) and the combination of trypsin-thermolysin hydrolysates (IC50 1.37 ± 0.84 mg/mL) had the highest antioxidant activity. The fractionation procedure with strong cation exchange (SCX) yielded fraction C from thermolysin hydrolysates, which exhibited the most significant antioxidant activity with an IC50 value of 0.47 ± 0.02 mg/mL. The fraction 4 derived from trypsin-thermolysin hydrolysates exhibited the most potent antioxidant activity (IC50: 0.34 ± 0.002 mg/mL). No previous reports have been made about the antioxidant properties of the hydrolysate fractions derived from C. formosanum proteins. Consequently, these findings could serve as a foundation for exploring peptides that have potential therapeutic sources for reducing oxidative stress.