Blue rot disease caused by Penicillium expansum is one of the most widespread fungal diseases that affects apples worldwide. This work was to verify the effect of chitosan (2 and 4 g/L) and its nano-form (0.2 and 0.4 g/L) against blue rot disease on apples and their effect on the expression of six defense-related genes as well as fruit quality parameters. Regarding disease incidence, in most cases, chitosan NPs performed better as compared to their raw materials for both artificial and natural infections. The highest efficacy was obtained for chitosan NPs at 0.4 g/L for artificial and natural infection in both 2019 and 2020 seasons. All treatments kept fruit quality parameters regarding firmness, total soluble solids, and titratable acidity for artificial and natural infection in both seasons. As expected, the exogenous application of chitosan NPs and bulk form triggered an increase in the expression levels of six defense-related genes including chitinase, peroxidase, β-1,3-glucanase, Xyloglucan endotransglycosylase (XET), pathogenesis-related protein (PR8), and phenylalanine ammonia lyase-1 (PAL1). Moreover, the highest mRNA quantity of all the studied genes was detected in leaves treated with chitosan NPs at both concentrations compared to other treatments. Chitosan NPs can be considered an eco-friendly and effective approach against blue mold of apples and can be integrated into management programs to maintain postharvest quality and extend the shelf life of fruits.