Compared to natural leather, microfiber synthetic leather has many excellent qualities, such as chemical resistance and physical and mechanical properties. However, preparation of microfiber synthetic leather with a high water vapor transmission rate (WVT), moisture absorption and wearing comfort property is still a challenge. In this study, we prepared thermoplastic polyurethane (TPU)/sulfonated polysulfone (SPSf) electrospun nanofibers and applied them to a microfiber synthetic leather base (MSLB). The effects of TPU/SPSf nanofiber content on the structure and properties of the MSLB were investigated. The results indicated that the TPU/SPSf nanofibers with an average diameter of 0.12 µm were well distributed at all directions in the MSLB. Differential scanning calorimetry analysis showed four Tg peaks, further demonstrating the existence of TPU/SPSf nanofibers. With the increase of TPU/SPSf nanofiber content from 0 to 30 wt%, the contact angles decreased gradually from 111.64° to 67.07°, leading to 55.19% improvement in the WVT value (from 2868.96 to 4452.24 g/(m2•24 h)) and 26.25% improvement in the moisture absorption (from 628.70% to 793.75% mm/s). Simultaneously, when the nanofiber content was 30 wt%, the nanofibers tended to bundle and 6.79% decrement of air permeability was observed. Specifically, the softness of the MSLB was improved by 88.55%. Moreover, the thermal stability and the tear strength were also obviously enhanced. Consequently, this research provided a feasible and promising way to prepare a high-performance MSLB using TPU/SPSf nanofibers.