Antioxidative activities were found in the culture supernatant of Serratia ureilytica TKU013 with squid pen as the sole carbon/nitrogen source. The 4th day supernatant showed the strongest antioxidant activities and the highest total phenolic content. The supernatant was further purified by liquid–liquid partition, and it was found that the ethyl acetate-soluble (EA) extract exhibited the strongest antioxidant activity in a DPPH radical-scavenging assay and this was compared with the positive control, α-tocopherol. This extract was further divided into eight fractions, designated as F1–F8, by silica gel liquid chromatography. In most cases, F4 and F8 were found to possess the strongest antioxidative activities. Significant associations between the antioxidant potency and the total phenolic content, as well as between the antioxidant potency and free amino groups, were found for the supernatant, extract, and fractions. With this method, squid pen waste can be utilized and has potential in the production of functional foods.