Influenced by the maturity and material properties, fresh corn has problems, such as low picking rate, high energy consumption and high damage rate during mechanized harvesting. For the above problems, a bionic reverse picking header was designed using the post-ripening morphology of corn and the hand-picking behavior as bionic prototypes. Model analysis and structural design of the key components of the header, including the reeling device, clamping device and picking device, were carried out. Based on the designed header prototype, single-factor tests and Box Behnken tests were conducted to explore the factors affecting the working performance of the picking header. The optimal structural parameters and working parameters were determined by response surface method. The results showed that with the increase in the clamping picking device speed and the stalk feeding speed, the picking rate first increased and then decreased. The interaction between feeding speed and cutter position had the most significant effect on the picking rate. The unique reverse picking mechanism and flexible device of the header could avoid collision and damage to the corn ears. The highest picking rate was achieved when the clamping picking device speed was 416.81 rpm, the stalk feeding speed was 1.13 m/s, and the cutter position was −5.45 cm.