Abstract: 　Objective　To explore the optimal storage standard of fresh human milk, and to observe the influence of different cold storage condition (time-temperature) on macronutrients (fat, protein, carbohydrates, TS and energy), immune substances (sIgA, lactoferrin, IL-6, 8, 10 and TNF-α) and bacteria indicators of fresh human milk. Methods　Fresh milk samples (n=30) were divided and stored at three temperature and nine time points, which are 4℃ (24 h, 48 h, 72 h), -18℃ (72 h, 7 d, 14 d, 4 w, 8 w, 12 w), and -80℃ (12 w, 24 w). At each time point, the macronutrients , immune substance, and bacteria colony counts of each milk sample were measured and compared with fresh milk. Results　Compared with fresh milk, all indicators with the exception of lactoferrin in stored human milk showed significant difference (P<0.05). Under 4℃ refrigeration condition, fat, IL-6, and TNF-α decreased, bacteria colony counts and Gram-positive colony counts increased over 72 h storage (P<0.05). Under -18℃ freezing condition, fat, protein, TS, energy and IL-6 decreased from 72 h to 12 w storage (P<0.05); carbohydrates and sIgA also decreased from 4 w and 8 w storage, respectively (P<0.05). Under -80 ℃ freezing condition, fat, protein, TS, energy and IL-6 decreased over 24 W storage (P<0.05). Conclusions　The macronutrients, immune substance, and bacteria indicators of human milk were affected obviously by cold storage. Refrigerated at 4℃ should not be longer than 72 h, -80℃ freezing condition should be chosen for more than two months storage.