Objective To establish a domestic ¹³C-octanoic acid breath test system for gastric emptying assessment, evaluate the abnomal gastric emptying and investigate the effect of gastroprokinetic agents on gastric empting in diabetic mice. Methods ¹³C-octanoic acid was synthesized with Grignard method. Diabetic mouse models were established by intravenous injection of alloxan tetrahydrate. After gastric irrigation with ¹³C-octanoic acid to mice, expired air samples were collected at various time points (0, 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105 and 120 min）and were examined with an infra red isotope analyzer for maximum concentration (Cmax) and half emptying time (T1/2) of ¹³CO₂. Effect of ¹³C-octanoic acid dosage on gastric emptying parameters and test reproducibility were analyzed. The effect of gastroprokinetic agent intervention on gastric empting of diabetic mice was evaluated, and the abnormal toxicity of ¹³C-octanoic acid was observed. Results ¹³C-octanoic acid dosage was positively related to Cmax (r=0.954，P<0.05), while had no significant effect on T1/2. The coefficient of variation for reproducibility was (4.76±1.43)%. There was no significant difference in T1/2 between normal group and diabetes group [(28.2±6.8) min vs (28.1±4.8) min, P>0.05]. Ghrelin significantly decreased T1/2 to (19.6±4.9) min （P<0.05）, while atropine significantly increased T1/2 to (40.7±7.0) min (P<0.05) in diabetic mice. No apparent toxicity was observed in ¹³C-octanoic acid abnormal toxicity test. Conclusion The established ¹³C-octanoic acid breath test system can evaluate the gastric emptying and determine the effect of gastroprokinetic agents on gastric empting in mice, with no invasion and favourable reproducibly.