The invention discloses a high-vitality cell preservation method for thalassiosira pseudonana. The high-vitality cell preservation method comprises the following steps: 1) inoculating microalgae in a culture medium so as to proliferate microalga cells to 10<6>-10<7> pieces/ml; 2) performing gradient cooling treatment on the cultured alga solution; 3) reducing the lighting intensity of lighting while cooling, wherein the reduction amplitude is 1000-2000lx/day; and 4) preserving the alga solution under a dark condition of 4-5 DEG C. The invention discloses the high-vitality cell preservation method for high-quality bait thalassiosira pseudonana, gradient cooling, light shielding and suspension alga solution high-vitality cell preservation are adopted, and the high-vitality cell preservation method is high in microalga survival rate, good in rejuvenation property, unlikely to cause pollution and simple, convenient and feasible to operate.