Previous studies have shown that calcium (Ca), magnesium (Mg), and Ca/Mg ratio are associated with inflammation and metabolic disorders, but their relationship with non-alcoholic fatty liver disease (NAFLD) is unclear. Thus, we aimed to explore the association between Ca, Mg, Ca/Mg ratio, and NAFLD in Chinese adults. We conducted a case-control study based on the Kailuan Cohort in China, including 1816 cases and 1111 gender- and age-matched controls. Dose-response relationships between blood Ca, Mg, Ca/Mg ratio, and NAFLD were evaluated using restricted cubic splines. Odds ratios (ORs) and their 95% confidence intervals (CIs) were estimated by logistic regression models. A negative association between blood Ca (overall association P < 0.001 and linear association P < 0.001) and NAFLD as well as Ca/Mg ratio (overall association P = 0.002 and linear association P = 0.024) and NAFLD was observed. Compared with the highest quartile, the adjusted OR (95% CI) for the lowest quartile of Ca and Ca/Mg ratio was 2.116 (1.679-2.667) and 1.358 (1.076-1.713), respectively. A U-shaped relationship was found for blood Mg and NAFLD, with the highest OR of 1.685 in the lowest quartile group when using the second quartile as a reference. Additionally, we observed the interaction between alanine aminotransferase and blood Ca (P = 0.024), total cholesterol (P = 0.017), low-density lipoprotein-cholesterol (P = 0.013), and blood Mg, as well as total cholesterol and Ca/Mg ratio (P = 0.014). Lower blood Ca and Ca/Mg ratio were significantly associated with the risk of NAFLD. Liver function or lipid metabolism parameters may modify their association, suggesting an individualized prevention strategy for NAFLD.