To evaluate the sub-chronic oral toxicity and maximum no observed adverse effect level (NOAEL) of L-alanine from fermentation broth in Sprague-Dawley (SD) strain rats.Methods According to procedures and methods for toxicological assessment of food (2003 edition), a total of 96 4-week-age cleaning grade weanling SD rats were divided randomly into 4 groups, 12 male rats and 12 female rats in each group. Rats were fed the diet containing 0.00,1.25,2.50 and 5.00 g/kg BW L-alanine from fermentation broth for 90 consecutive days respectively. On the 45^th day, blood samples collected from inner canthus veniplex were used for complete blood count. At the end of the experiment, samples of blood were collected for hematological and biochemical analysis, respectively. Livers, spleens, kidneys and testicles (ovariesy) from all rats were weighed, furthermore, and the ratio of these organs to body weight was determined. The brain (including cerebrum, cerebellum and pons cerebelli), heart, thymus, lung, liver, kidneys, adrenal gland, spleen, stomach, duodenum, testicles, ovariesy and uterus (horn and cervix) were removed for histopathological examinations. Results The growth and development of all rats in four groups generally performed well with normal growth and development. There were no significant differences between three experimental groups and the control group (P>0.05), including the final body weight, total food intake, total food utilization, the cardinal organs/tissues’ wet weight and organ coefficient. No sample-related significant changes were observed on haematology and serum biochemistry parameters at the mid or final term. Histopathological examinations showed no damage caused by L-alanine from fermentation broth, except for a few of the common spontaneous lesions.Conclusion The NOAEL of male and female SD rats in oral sub-chronic toxicity study for L-alanine from fermentation broth were 4.58,5.56 g/kg BW respectively.