Population-based seroprevalence of chronic hepatitis B and C infections has not been examined in Lao People’s Democratic Republic (PDR). Therefore, this study aimed to estimate the seroprevalence of these infections in the general population of Lao PDR and perform subgroup analysis. A nationwide seroprevalence survey was conducted in Lao PDR in June 2019 using the multistage cluster sampling method. Dried blood spot samples were collected onto WhatmanTM 903 filter paper by finger prick. A chemiluminescent microparticle immunoassay was used to measure the levels of hepatitis B surface antigen (HBsAg) and hepatitis C antibody (HCV-Ab). Samples in which the HBsAg level was above 0.05 IU/ml and HCV-Ab was above the signal/cutoff ratio of 1.0 were considered positive based on comparisons with the relative light unit value of a calibration sample. A total of 1,927 samples (male: 47.3%, mean age: 23.0 years) were included in the analysis. The prevalence was estimated to be 4.2% (95% confidence interval [CI]: 2.7–6.3) for HBsAg and 1.6% (95% CI: 0.5–5.3) for HCV-Ab. Multivariable analysis revealed that those aged 20–24 years (adjusted odds ratio (AOR): 2.3, 95% CI: 1.1–4.6), those aged 25–29 years (AOR: 2.7, 95% CI: 1.3–5.6), those from the Northern region (AOR: 2.8, 95% CI: 1.2–6.6), and those who were Khmu (AOR: 3.6, 95% CI: 2.0–6.8) or Hmong (AOR: 5.0, 95% CI: 3.3–7.5) were significantly more likely to be positive for HBsAg. Although there were no statistically significant differences in the HCV-Ab prevalence according to each variable, males (2.9%, 95% CI: 0.7–10.7), those aged ≥40 years (6.1%, 95% CI: 2.1–16.8), and those from the Southern region (3.3%, 95% CI: 0.6–15.3) tended to have a higher prevalence. This novel population-based survey found differences in the prevalence of chronic hepatitis B and hepatitis C virus infections in Lao PDR according to sex, age group, region, and ethnicity; however, the results of this study should be confirmed in future studies, and relevant responses tailored for each target also need to be determined to control the transmission of hepatitis B and C infections.