Until now, most studies using calcium alginate gel (CAG) have been conducted primarily at room temperature (20 °C) without considering gelation temperature. Moreover, the effects of gelation temperature on the physical properties of CAG beads have not been studied in detail. We aimed to study the effect of gelation temperature on the physical properties (diameter, sphericity, and rupture strength) of CAG beads. Response surface methodology was used in this study. The independent variables were sodium alginate concentration (X1, 1.2–3.6%, w/v), calcium lactate concentration (X2, 0.5−4.5%, w/v), gelation temperature (X3, 5–85 °C), and gelation time (X4, 6–30 min). Diameter (Y1, mm), sphericity (Y2, %), and rupture strength (Y3, kPa) were selected as the dependent variables. A decrease in gelation temperature increased the diameter, sphericity and rupture strength of the CAG beads. Additionally, the CAG beads prepared at 5 °C exhibited the highest rupture strength (3976 kPa), lowest calcium content (1.670 mg/g wet), and a regular internal structure. These results indicate that decreasing the gelation temperature slows the calcium diffusion rate in CAG beads, yielding a more regular internal structure and increasing the rupture strength of the beads.