Abstract: Pleurotus ostreatus has been one of the most widely cultivated and the largest edible fungi in China. Global production has been growing rapidly, due to simple cultivation, low cost, quick effect, high yield, and short cycle. According to the statistics of the China Edible Mushroom Association, the output of Pleurotus ostreatus in 2020 was 6.8647 million tons, accounting for 17.45% of the total output of edible fungi, indicating the third-largest cultivar in China. However, there is still a great challenge on the efficiency and stability in the current production of oyster mushroom cobs combined with the induction, sterilization, and fermentation of Pleurotus ostreatus cultivation material and the central-hole inoculation. Taking a corncob particle as the Pleurotus ostreatus fermentation material, this study aims to develop a strain charging and inoculating machine, particularly for the higher efficiency of single machine operation, high stability, and full automation. Three devices were mainly composed of a charger, a nest, and an inoculation device. The key components of the whole machine were designed, according to the requirements of the charging quality and inoculation amount in the production of bacterial sticks, thereby determining the structural parameters of the charging auger, the socket groove cam, and the inoculation cam. A response surface method was used to optimize the test factors, where the diameter of the auger and the pitch of the auger were the influencing factors, and the charging productivity and density were the response indicators. A regression mathematical model was established for the charging productivity and density. The response value of the rotation speed of the auger was obtained to determine the most significant impact. Furthermore, the inoculum amount of bacteria was measured under different inoculation electromechanical speeds. As such, the inoculum amount was 132.33 g, and the inoculation variation coefficient was 1.19 %, when the inoculation motor speed was 120 r/min, which satisfied the number of bacteria in the center hole of the fermented material. The production requirement was measured to be 242.5 mm less than 5% of the charge and the average inoculation depth of the inoculation unit. Multi-objective optimization of the regression model was carried out to further verify the charging performance of the equipment. The optimal structural parameters of the charging device were obtained as follows: the auger rotation speed of 191.94 r/min, auger diameter 133.22 mm, auger pitch 100.00 mm, while the charging productivity was 723 bags/h, the charging density was 537.27 kg/m3, and the charging mass was 2.42 kg. The optimized all-in-one and stand-alone machine was verified for production. It was found that the charging productivity of the all-in-one machine was 726 bags/h, the charging density was 536.25 kg/m3, the coefficient of variation of the charging quality was 0.82%, and the coefficient of variation of the inoculation amount was 0.92%, the coefficient of variation of the inoculation depth was 1.05%, the mouth of the fungus bag was firm and flat, while the folds were uniform, and the nest has no leaking bags. This key component can fully meet the equipment demand for the mechanized production of oyster mushroom fermentation materials in northern regions.