The demands for long span structures with reduction of story height have greatly increased as building structures become much larger and higher in recent years. Although the development of flexural members for reducing story height or realizing long span has been studied by many researchers and engineers, there is still a lack of efficient systems that meet these two demands simultaneously. Thus, this study proposed a prestressed composite girder with corrugated web suitable for long span and reduction of story height. The corrugated webs utilized in this development make the accordion effect introducing the larger effective prestressing force to top and bottom flanges, which causes the larger upward camber reducing the member deflection. The concepts of effective moment of inertia and effective web area is proposed to reflect the accordion effect of the corrugated web before composite. Five full-scaled specimens, with key variables of prestressing, welding length of web, the number of drape points, and the use of shear connectors, were tested to investigate their flexural behavior and to verify the performance of the proposed composite girder. The test results verified that the proposed prestressed composite girder had an excellent flexural performance with greate flexural strength and stiffness.