To address the challenges of labor-intensive and costly manual oyster spat insertion in longline-suspended farming, an automated oyster spat insertion device was designed based on negative pressure suction and bundling fixation technologies. Using this device as the experimental platform, a three-factor, three-level Box–Behnken experiment was conducted, with fixation mechanism inclination, negative pressure suction cup span, and horizontal distance between turnover and fixation mechanisms as the experimental factors. The performance of the device was evaluated using the effective fixation rate and damage rate as the experimental indicators. The quadratic polynomial regression models were established to assess the impact of these factors on operational performance, while the response surface method was employed to analyze the interaction effects between factors. Parameter optimization and experimental validation were also performed. The results indicate that the factors affecting the effective fixation rate, in order of significance, are as follows: fixation mechanism inclination, horizontal distance between turnover and fixation mechanisms, and negative pressure suction cup span. For the damage rate, the order of significance is as follows: fixation mechanism inclination > negative pressure suction cup span < horizontal distance between turnover and fixation mechanisms. The optimization results show that when the fixation mechanism inclination is set at 43°, the negative pressure suction cup span at 27 mm, and the horizontal distance between turnover and fixation mechanisms at 179 mm, the effective fixation rate reaches 92.08%, and the damage rate is 4.71%. The relative errors between the measured and model-predicted values are less than 5%, indicating that the regression models are reliable. This research provides valuable insights for advancing the mechanization of the oyster farming industry and replacing manual labor with mechanized equipment.