Stepwise structural evolution of nanofibers through electrospinning was captured by AFM imaging. Nanofiber evolution exhibited five characteristic structures in turn: columns, plates, micropores, bumps and smooth surface. Based on our results and previous studies, these five stages are discussed in detail and the mechanism of nanofiber formation is proposed. In addition, the drug incorporation process of blend-electrospun nanofibers was shown vividly and clearly by AFM. It suggested that drugs could be completely incorporated into nanofibers by blend-electrospinning if proper polymers and drugs were chosen. Furthermore, the effects of solution properties on nanofiber structural evolution were also studied systematically.