Squid chondroitin sulfate samples CS-1 (molecular weight: 39,800 u) and CS-2 (molecular weight: 19,500 u) were used as the raw materials in this study, and the relationships between molecular weight and moisture retention capacity, antioxidant activity, and ability to inhibit melanoma growth were explored. The results indicated that the moisture retention capability of squid chondroitin sulfate was weaker than that of glycerin, and the higher the molecular weight, the stronger the moisture retention capability. The half-maximal inhibitory concentration (IC50) values of CS-1 and CS-2 in scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH?)?radicals were 13.64 and 12.42 mg/mL, respectively, and the scavenging rate of pure product was higher than that of crude product. The IC50 values of CS-1 and CS-2 in scavenging ?OH radicals were 7.52 and 3.98 mg/mL, respectively, and the scavenging abilities were in the following order: CS-2 > crude > CS-1. Strong antioxidant activity was displayed by the squid chondroitin sulfate. The indirect measurement of the reducing power on Fe3+ also showed that smaller molecular weight resulted in stronger antioxidant activity. The results suggested that both CS-1 and CS-2 could inhibit the growth of B16-F10 melanoma cells, and significant inhibitory effect was seen at a concentration of 120 μg/mL. When the concentration was 240 μg/mL, the tumor inhibition rate of CS-2 was higher than that of CS-1. The smaller the molecular weight, the stronger the antitumor activity. The inhibition rate of tumor cells by CS-2 was higher than the IC50, reaching 54%; under the same conditions, the inhibition rate by cisplatin (CDDP) was 79%, and therefore, CS-1 and CS-2 have significant antitumor activity.