Acute-on-chronic liver failure (ACLF) is a syndrome characterized by acute decompensation, organ failures, and high short-term mortality. Hepatitis B virus (HBV) is the main cause of liver failure in China. Urinary tract infection (UTI) is one of the common bacterial infections in patients with HBV-ACLF. However, few studies concerning the risk factors and epidemiology have been published. A retrospective analysis of 539 patients with HBV-ACLF was performed. The prevalence, bacterial profile, and antibiotic susceptibility pattern were investigated and associated risk factors of UTI in patients with HBV-ACLF were evaluated with a logistic regression model. The overall prevalence of UTI among the study participants was 26.53% (143/539), and 64.34% (92/143) of them were asymptomatic. One hundred thirty-five strains of bacteria, including 74.07% (100/135) gram-negative bacteria and 53.33% (72/135) multidrug-resistant organisms, were cultivated from 143 patients with HBV-ACLF. Escherichia coli 46.67% (63/135) and Klebsiella pneumoniae 13.33% (18/135) were the most common bacteria. The antibiotic susceptibility test pattern showed that 92.93%, 81.63%, and 81.63% of the gram-negative isolates were sensitive to imipenem, tigecycline, and piperacillin/tazobactam, respectively. Meanwhile, all the gram-positive isolates were sensitive to linezolid, teicoplanin, and vancomycin. Compared with non-UTI group, the patients with UTI had higher serum creatinine, lower educational status, total bilirubin, direct bilirubin, and albumin. Finally, educational status and albumin were independent risk factors in the prevalence of UTI in patients with HBV-ACLF. UTI is one of the common bacterial infections seen in patients with HBV-ACLF. Gram-negative bacteria account for the majority of cultured bacteria, and multidrug-resistant bacteria are common. UTI is determined by a diverse set of complex factors, which lower educational status and hypoalbuminemia predict the more prevalence of UTI.