In this study, a total 1737 bp ascorbate oxidase (AO) gene encoding a 578 amino acid protein was cloned from strawberry (Fragaria×ananassa cv. Toyonaka). Real-time quantitative expression analysis was conducted on FaAO gene of strawberry fruits during 7 developmental stages and in 5 strawberry tissues. The results revealed that during 7 fruit developmental stages, FaAO expressing level gradually increased in yonger stages, and declined dramatically in maturing stages, in which it could barely be detected. According to experimental results, we can conclude that FaAO expresses at a higher level in younger fruits, and lower in latter stages. This could be related to the fact that younger fruits are growing actively, and FaAO has a significant effect on facilitating fruit growth. Results for expression in 5 tissues indicated the highest expression was detected in flowers, and the lowest in fruit tissue (at full red stage). One of the possible reasons is that intense respiration and high ethylene synthesis occur in the mature fruits, repressing the expression of FaAO gene in strawberry fruits. Also, it could be a reason why the ascorbic acid (AA) content in mature fruits is higher than that in other tissues and stages. The differences of expression results among 7 developmental stages and 5 tissues indicated a novel expression pattern of FaAO. We propose that FaAO is correlated with strawberry growth and development, the depression of the FaAO expression can lead to apparent accumulation of AA in strawberry plant.