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Leaf spot disease caused by Lasiodiplodia theobromae is one of the most serious fungal diseases of kiwifruit production. In this work, the co-application of isopyrazam·azoxystrobin and chitosan against leaf spot disease in kiwifruit and its effects on disease resistance, photosynthesis, yield, quality, and amino acids of kiwifruit were investigated. The results show that isopyrazam·azoxystrobin exhibited a superior bioactivity against L. theobromae with an EC₅₀ value of 0.1826 mg kg⁻¹. The foliar application of chitosan could effectively enhance isopyrazam·azoxystrobin against leaf spot disease with a field control efficacy of 86.83% by spraying 29% isopyrazam·azoxystrobin suspension concentrate (SC) 1500 time + chitosan 100-time liquid, which was significantly (p < 0.05) higher than 78.70% of 29% isopyrazam·azoxystrobin SC 1000-time liquid. The co-application of isopyrazam·azoxystrobin and chitosan effectively enhanced soluble protein, resistance enzymes’ activity in kiwifruit leaves, and reduced their malonaldehyde (MDA), as well as reliably improved their photosynthetic characteristics. Simultaneously, their co-application was more effective in promoting growth, quality, and amino acids of kiwifruit fruits compared to isopyrazam·azoxystrobin or chitosan alone. This study highlights that the co-application of isopyrazam·azoxystrobin and chitosan can be used as a green, safe, and efficient approach for controlling leaf spot disease of kiwifruit and reducing the application of chemical fungicides. Full article