After suffering from mechanical injury and fungal infection, grapes are perishable. Botrytis cinerea, the causal agent of gray mould, is a critical pathogen for grapes. In this study, the inhibitory effect of Pseudomonas fluorescens on the formation of gray mould on grapes during the postharvest storage was investigated on “Kyoho” grape. The results suggest that a living cell suspension of P. fluorescens significantly inhibited spore germination of B. cinerea, and significantly reduced the incidence of grape gray mould. Moreover, compared with the control, the fruit inoculated with P. fluorescens had elevated activities of polyphenol oxidase (PPO), peroxidase (POD), catalase (CAT), phenylalanine ammonia-lyase (PAL), chitinase (CHI), and β-1,3-glucanase (GLU). Increase in enzyme activity correlated with enhanced host resistance. In addition, there was little difference in storage quality between the treated group and control group, indicating no adverse effects of the induced defence response on fruit quality.