The effects of single/combined administration of 9 probiotics on ammonia (NH3) emissions during in vitro fermentation of the caecal contents of laying hens were studied. Not all of the probiotics reduced NH3 emissions. Pichia farinose, Bacillus coagulans, Lactobacillus plantarum, Pichia guilliermondii, and Bacillus subtilis reduced NH3 production by approximately 35.1 to 39%. Compared with the control group, the greatest NH3 inhibition was achieved via the combined application of P. guilliermondii, B. subtilis, and L. plantarum at 1:2:1, resulting in a 46% reduction. Effective probiotics use decreased crude protein digestibility, pH, ammonium nitrogen, valerate levels, and urease and uricase activity, but increased urea, purine trione, nitrate nitrogen, total volatile fatty acids, and acetate levels. The relative abundance of the bacteria responsible for fermenting carbohydrates to produce short fatty acids was increased. Under different treatments, the KEGG Orthology (KO) metabolic pathways of NH3 production and utilization were diverse. Hence, the application of probiotics to control NH3 emissions is dependent on the types and combined ratio of the organisms involved.