Objective To investigate the association of alpha-fetoprotein （AFP） and prealbumin （PAB） with the 90-day prognosis of patients with hepatitis B virus-related acute-on-chronic liver failure （HBV-ACLF），as well as the difference in 90-day prognosis between the patients with different levels of AFP and PAB. Methods A total of 371 HBV-ACLF patients who were hospitalized in The General Hospital of Western Theater Command from January 2018 to January 2023 were enrolled，and according to the follow-up results on day 90 after discharge，they were divided into survival group with 216 patients and death group with 155 patients. The medical record system was used to collect general data，AFP，PAB，and other related laboratory markers. The t-test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups，and the least significant difference t-test was used for comparison between two groups. The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups，and the Kruskal-Wallis H test was used for comparison between multiple groups and further comparison between two groups. The chi-square test was used for comparison of categorical data between groups. The multivariate logistic regression analysis was used to identify the influencing factors for the prognosis of HBV-ACLF patients. The receiver operating characteristic （ROC） curve was plotted for AFP and PAB to determine their cut-off values. The Kaplan-Meier method was used to plot survival curves，and the Log-rank test was used for comparison. Results Compared with the death group，the survival group had significantly higher levels of hemoglobin （Hb），PAB，AFP，and platelet count （PLT）（all P<0.05） and significantly lower age，total bilirubin （TBil），white blood cell count （WBC），cystatin，creatinine，urea，international normalized ratio （INR），Model for End-Stage Liver Disease （MELD） score，proportion of patients with Child-Pugh class C，and incidence rates of ascites and hepatic encephalopathy （all P<0.05）. The multivariate logistic regression analysis showed that PAB （odds ratio ［OR］=0.985，95% confidence interval ［CI］：0.972‍ ‍—‍ ‍0.998，P=0.024），AFP （OR=0.998，95%CI：0.996‍ ‍—‍ ‍1.000，P=0.028），PLT （OR=0.989，95%CI：0.982‍ ‍—‍ ‍0.996，P=0.003），age （OR=1.046，95%CI：1.018‍ ‍—‍ ‍1.075，P=0.001），TBil （OR=1.004，95%CI：1.002‍ ‍—‍ ‍1.006，P<0.001），and WBC （OR=1.237，95%CI：1.110‍ ‍—‍ ‍1.379，P<0.001） were independent influencing factors for 90-day prognosis in HBV-ACLF patients. According to the cut-off values of AFP and PAB on ROC curves，the patients were divided into group A with 102 patients （AFP≥73.19 ng/mL and PAB≥22.55 mg/L），group B with 170 patients （AFP≥73.19 ng/mL and PAB<22.55 mg/L； AFP<73.19 ng/mL and PAB≥22.55 mg/L），and group C with 99 patients （AFP<73.19 ng/mL and PAB<22.55 mg/L）. There were significant differences between these three groups in age，Hb，INR，MELD score，and Child-Pugh class （all P<0.05）. The Kaplan-Meier survival analysis showed that group A had a significantly higher 90-day cumulative survival rate than groups B and C （χ²=19.825，P<0.001）. Conclusion AFP combined with PAB can better predict the 90-day prognosis of HBV-ACLF patients，and patients with high levels of AFP and PAB tend to have a lower mortality rate on day 90.