Higher intakes of fish and n-3 polyunsaturated fatty acids (n-3PUFAs: eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) are associated with a reduced risk of coronary artery disease (CAD). We investigated the relationships between fish-derived n-3PUFAs and prevalence of CAD, and to assess the association of n-3PUFAs with atherosclerosis-related markers in an urban area. This study was designed as a hospital-based cross-sectional study on 649 consecutive outpatients who had undergone regular examinations between April 2009 and October 2009. After adjustments for the coronary risk factors in a multilogistic regression analysis of variables for which a significant difference was identified between the group of patients with a prevalence of CAD and the group with no prevalence of CAD, the multivariable odds ratio (95 % confidence interval) was 0.394 (0.205/0.760; P = 0.005) for the highest (92.4–373.5 μg/ml) versus lowest (6.2–40.0 μg/ml) quartile of serum EPA values and 0.433 (0.228/0.824; P = 0.011) for the highest (160.7–451.8 μg/ml) versus lowest (35.7–100.7 μg/ml) quartile of serum DHA values. Multivariate regression analyses after adjustment for risk factors showed that higher serum EPA and DHA levels were independent variables of a higher level of serum apolipoprotein A-1, a major compound of high-density lipoprotein. However, the results suggested that there might be conflicting effects of EPA and DHA in regard of the serum levels of other lipid markers. This cross-sectional study suggests that higher serum levels of n-3PUFAs were associated with a lower prevalence of CAD and an increase in serum apolipoprotein A-1 level, even in an urban area.