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In-line pesticide mixing technology offers an effective solution to challenges such as health threats to operators, environmental pollution, and the wastage of pesticide solutions associated with traditional premix methods. Focusing on the design of a static mixer intended for use in an in-line pesticide mixing system, to verify optimal performance, the mixing characteristics of the in-line flow field were investigated through computational fluid dynamics (CFD) simulations. Single-factor test, Plackett-Burman test, and Box-Behnken center combination test were employed to evaluate the static mixer pressure drop and mixing uniformity. The impacts of parameters such as tilt spoiler angle, center through-hole diameter, mixing unit spacing, and flow-through hole diameter on the evaluation indexes were analyzed by using the response surface method. On this basis, multi-objective optimization, targeting low-pressure drop and high mixing uniformity was conducted through a genetic algorithm. The optimal structural parameters were determined as follows: spoiler inclination of 55.38°, mixing unit spacing of 22.64 mm, and pore size of 1.64 mm. Bench tests for pressure drop and mixing uniformity were conducted under specific conditions: electric control valve opening for liquid pesticide injection at 33%, continuous operation of the pesticide pump, and variable working gears for the water pump. The results indicated an increasing trend in pressure drop within the Reynolds number range of 5 984~13 286 and the pressure drop factor was less than 106.7. The coefficient of variation for mixing uniformity exhibited a decreasing trend, and it was less than 0.008 5, indicating the reliable performance of the static mixer. The research results can provide reference for the development and structure optimization of static mixer in China.