In order to improve the clinical diagnosis efficiency of bovine respiratory disease complex (BRD) based on coinfection of multiple pathogens, this study established a real-time quantitative PCR (qPCR) to multiple and rapid detection for common seven pathogens of BRD including Mycoplasma bovis (M.b), Pasteurellae multocida (P.m), Mannheimia haemolytica (M.h), Bovine infectious rhinotracheitis virus (IBRV), Bovine syncytial virus (BRSV), Bovine parainfluenza virus type 3 genotype a (BPIV-3a) and c (BPIV-3c), the specific primers and TaqMan probes were designed for the oppD/F gene of M.b, gcp gene of M.h, ompH gene of P.m, gB gene of IBRV, N genes of BRSV, BPIV-3a and BPIV-3c and synthesized. After optimization of reaction conditions, a multiple qPCR was established for simultaneous detection of the above 7 pathogens in three tubes. This method specifically amplified these 7 pathogens, rather than other main pathogens common in cattle, indicating a high specificity. For M.b, P.m, M.h, IBRV, BRSV, BPIV-3a and BPIV-3c, the analytical sensitivity as limit of detection (LOD) was 102 copies /μL, 102 copies /μL, 101 copies /μL, 102 copies /μL, 102 copies /μL, 102 copies /μL, 102 copies /μL and 101 copies /μL, respectively, suggesting a high sensitivity. In addition, the coefficient of variation of the method within the group and between the groups were less than 2.5% and 5.5%, respectively, indicating a good repeatability. Furthermore, 115 clinical nasal swabs were parallelly detected by this method and conventional PCR, and the positivity was 27.83% for M.b, 36.65% for P.m, 25.22% for M.h, 11.30% for IBRV, 0.95% for BRSV, 8.57% for BPIV-3C. In addition, the proportion of co-infection was 26.1% with 11 patterns of pathogen combinations. Among them, coinfection of M.b with other pathogens ranked the top 1 with the proportion of 72.7% (8/11). Within the 30 coinfection cases, M.b/P.m combination mostly occurred with 60% (18/30); the top three pathogens present in the coinfection was M.b, P.m, and M.h with frequency of 73.3% (22/30), 73.3% (22/30) and 43.3% (13/30); followed by IBRV (26.7%, 8/30) and BPIV-3c (13.3%, 4/30). Overall, this method has a high sensitivity and specificity, and a potential application in clinical detection of single pathogen and multi-pathogen infection causing BRD.