Seasonal changes in the biochemical composition of Hemicentrotus pulcherrimus roe were analyzed from May 2016 to February 2017 in Korea. Spawning, occurring between April and May, was characterized by relatively low lipid (3.46 g/100 g) and protein levels (13.22 g/100 g). Concurrently, calcium level was increased, while magnesium level was decreased. The roe’s polyunsaturated fatty acids (PUFA) content was high, constituting 40% of total fatty acids, with eicosapentaenoic acid (C20:5n-3) and eicosatrienoic acid (C20:3n-3) being the most predominant. During the spawning season, lower levels of ascorbic acid (30.9 mg/kg) and folic acid were observed alongside higher concentrations of riboflavin (7.24 mg/kg) and niacin (232.32 mg/kg). Vitamin E content was at its lowest in May, measuring 0.05 mg/100 g, compared to a range of 1.62–2.22 mg/100 g in other seasons. Overall, the results suggest that H. pulcherrimus roe is a valuable source of protein, minerals, vitamins, and glycine, an important flavor component. However, significant variations in its biochemical components were noted during the spawning period compared to other seasons.