In this paper, we synthesized amphiphilic chitosan using one-pot method with the goal to develop delivery system for the natural pesticide spinosad. Chitosan-Isoleucine-Polyethylene glycol methyl ether (CIP) copolymers based nanomicelles (CIPNMs) were prepared by self-assembly with the assistance of ultrasonic treatment and then the spinosad was loaded on the micelles. Sphere-shaped spinosad-loaded CIPNMs (SSD@CIPNMs) with the particle sizes of approximately 454.9 nm were obtained with the CIP co-polymers. The shell of CIPNMs could effectively prevent the photodegradation of spinosad and played a role of protective barrier. In addition, the prepared SSD@CIPNMs not only delayed the release of spinosad but also had a property of pH-responsive. The release rate of SSD@CIPNMs at pH = 6.4 was greater than that of at pH = 7.0. And, the contact angle of SSD@CIPNMs on banana leaves surface was 60.47 degrees, which meant SSD@CIPNMs had a good affinity with banana leaves. Furthermore, SSD@CIPNMs showed higher antifungal activity against Fusarium oxysporum compared with the unencapsulated spinosad at the same drug dose. This work was contribution to the development of nano-drug carrier systems of natural polymer materials, with a great potential for targeted treatment of banana wilt.