The non-uniformity effect of the inter-foil distance has been studied using a gaseous electron multiplication (GEM) detector with sensitive area of 50mm × 50mm. A gradient of the inter-foil distance is introduced by using spacers with different heights at the two ends of the foil gap. While the cluster size and the intrinsic spatial resolution show insignificant dependence on the inter-foil distance, the gain exhibits an approximately linear dependence on the inter-foil distance. From the slope, a quantitative relationship between the change of the inter-foil distance and the change of the gain is derived, which can be used as a method to evaluate the non-uniformity of the foil gap in the application of large-area GEM detectors.