The aim of this study was to identify the target of nonalcoholic fatty liver disease (NAFLD) cell-specific aptamer NAFLD01 and investigate its effect on lipid metabolism in vitro. A distinct membrane protein of NAFLD cells pulled down by NAFLD01 was analyzed by mass spectrometry to determine target candidates, and affinity of NAFLD01 to target-protein-silent NAFLD cells was detected to validate it. Knockdown of CD36 abolished the binding of NAFLD01, and its binding affinity was associated with membrane-bound CD36. NAFLD01 affinity for NAFLD cells was proportional to the CD36 expression level. Moreover, compared to random sequences, NAFLD01 showed better recognition for both mouse and human tissue sections of NAFLD. Importantly, NAFLD01 could ameliorate liver fat deposition through interaction with CD36 in vitro. Therefore, aptamer NAFLD01 could act as an effective and safe targeted drug for NAFLD. NAFLD01 is the first reported CD36-specific aptamer. This aptamer can improve hepatocyte steatosis via specifically binding to CD36. This study provides a molecular tool to investigate the mechanism of CD36 in NAFLD.