In order to investigate the pollution risk of multidrug-resistant bacteria in biogas residue from anaerobic digestion of cattle manure, five antibiotics (erythromycin, ampicillin, chlortetracyline, streptomycin and ciprofloxacin) that commonly used in livestock and poultry breeding were used to screen the culturable antibiotic-resistant bacteria from biogas residue of cattle manure. The results showed that the proportion of multidrug-resistant bacteria was as high as 76.5%, with 11 and 21 strains of bacteria resistant to five and four antibiotics, respectively. All multidrug-resistant bacteria were resistant to ampicillin, and bacteria resistant to erythromycin, chlortetracyline and streptomycin accounted for 92.0%, 89.3% and 61.3% of the total multidrug-resistant bacteria count, respectively. Through bacterial 16S rRNA sequencing, 32 strains with more than four kinds of resistance were identified as Shigella flexneri, Morganella morganii and Ochrobactrum pseudointermedia, respectively, all of which were important clinical pathogens. Resistance genes carried by seven typical multi-drug resistant bacteria strains were analyzed using whole genome sequencing, and a total of 28 antibiotic resistance genes were detected, corresponding to nine antibiotic resistance types. Analysis of the antibiotic resistance genes and insertion sequences locus information revealed that multidrug-resistant bacteria generally carried plasmids containing both resistance genes and insertion sequences, indicating that these resistance genes had high mobility and strong transmission risk. In conclusion, the biogas residue of cow manure contains a large number of multidrug-resistant bacteria, which can be regarded as a reservoir of antibiotic resistance genes, and attention should be paid to the environmental risk monitoring and assessment during the agricultural resource utilisation of biogas residue.