NH3 emission has become one of the key factors for aerobic composting of animal manure. It has been reported that adding microbial agents during aerobic composting can reduce NH3 emissions. However, environmental factors have a considerable influence on the activity and stability of the microbial agent. Therefore, this study used cornstalk biochar as carriers to find out the better biological immobilization method to examine the mitigation ability and mechanism of NH3 production from laying hen manure composting. The results from different immobilized methods showed that NH3 was reduced by 12.43%, 5.53%, 14.57%, and 22.61% in the cornstalk biochar group, free load bacteria group, mixed load bacteria group, and separate load bacteria group, respectively. Under the simulated composting condition, NH3 production was 46.52, 38.14, 39.08, and 30.81 g in the treatment of the control, mixed bacteria, cornstalk biochar, and cornstalk biochar separate load immobilized mixed bacteria, respectively. The cornstalk biochar separate load immobilized mixed bacteria treatment significantly reduced NH3 emission compared with the other treatments (p < 0.05). Compared with the control, adding cornstalk biochar immobilized mixed bacteria significantly decreased the electrical conductivity, water-soluble carbon, total nitrogen loss, and concentration of ammonium nitrogen (p < 0.05), and significantly increased the seed germination rate, total number of microorganisms, and relative abundance of lactic acid bacteria throughout the composting process (p < 0.05). Therefore, the reason for the low NH3 emission might be due not only to the adsorption of the cornstalk biochar but also because of the role of complex bacteria, which increases the relative abundance of lactic acid bacteria and promotes the acid production of lactic acid bacteria to reduce NH3 emissions. This result revealed the potential of using biological immobilization technology to reduce NH3 emissions during laying hen manure composting.