Due to the innate extracellular matrix mimicking features, fibrous materials exhibited great application potential in biomedicine. In developing excellent fibrous biomaterial, it is essential to reveal the corresponding inherent fiber features' effects on cell behaviors. Due to the inevitable “interference” cell adhesions to the background or between adjacent fibers, it is difficult to precisely reveal the inherent fiber diameter effect on cell behaviors by using a traditional fiber mat. A single-layer and parallel-arranged polycaprolactone fiber pattern platform with an excellent non-fouling background is designed and constructed herein. In this unique material platform, the “interference” cell adhesions through interspace between fibers to the environment could be effectively ruled out by the non-fouling background. The “interference” cell adhesions between adjacent fibers could also be excluded from the sparsely arranged (SA) fiber patterns. The influence of fiber diameter on stem cell behaviors is precisely and comprehensively investigated based on eliminating the undesired “interference” cell adhesions in a controllable way. On the SA fiber patterns, small diameter fiber (SA-D1, D1 means one μm in diameter) may seriously restrict cell proliferation and osteogenesis when compared to the middle (SA-D8) and large (SA-D56) ones and SA-D8 shows the optimal osteogenesis enhancement effect. At the same time, the cells present similar proliferation ability and even the highest osteogenic ability on the densely arranged (DA) fiber patterns with small diameter fiber (DA-D1) when compared to the middle (DA-D8) and large (DA-D56) ones. The “interference” cell adhesion between adjacent fibers under dense fiber arrangement may be the main reason for inducing these different cell behavior trends along with fiber diameters. Related results and comparisons have illustrated the effects of fiber diameter on stem cell behaviors more precisely and objectively, thus providing valuable reference and guidance for developing effective fibrous biomaterials.