In order to explore the effects of ozonated water treatment on the initial microflora of fresh grass carp fillets from different sources， fresh grass carp fillets at different sampling times were used as raw materials to determine the total viable count (TVC)， thiobarbituric acid (TBA)， pH value， chroma and sensory score， and to study the effects of different parameters of ozonated water treatment on the physicochemical properties of fresh grass carp fillets， so as to optimize the treatment conditions. The optimized conditions were used to treat fresh grass carp fillets， and the changes in the microflora of fresh grass carp fillets before and after ozonated water treatment were investigated using traditional isolation culture combined with 16S rRNA identification and high-throughput sequencing. The results showed that with the increase of ozonated water treatment mass concentration and treatment time， the number of residual bacteria and the rate of bacterial reduction of fresh grass carp fillets improved significantly and the microflora changed significantly after treatment (P<0.05). Ozonated water treatment mass concentration and treatment time had no significant effect (P>0.05) on TBA and pH of fresh grass carp fillets， but had a significant effect (P<0.05) on colour values. In terms of sensory scores， ozonated water treatment mass concentration had no effect， but treatment time had a significant effect and sensory quality decreased significantly with extended treatment time. After treatment of fresh grass carp fillets with 15 mg/L ozonated water spray for 5 min， the bacterial species of the three different sampling time of fresh grass carp fillets decreased by 52.17%， 50.00% and 60.00%， respectively， the bacterial diversity decreased significantly (P<0.05) and the proportion of Gram-positive (G+) bacteria increased. The results of the principal co-ordinates analysis showed that the degree of similarity in the microflora of grass carp fillets from different sampling time increased after ozonated water treatment. In conclusion， the treatment of raw grass carp fillets with 15 mg/L ozonated water for 5 min resulted in a reduction rate of 80.66%， and the microflora of fresh grass carp fillets at different sampling time became simple and similar after the treatment.