The demand for personal protective equipment increased exponentially during the COVID-19 epidemic, and the shortage of medical protective clothing has prompted consideration for the recycling of medical protective clothing. However, it is still challenging to maintain an excellent barrier property and functionality for medical protective material after cyclic sterilization. Herein, the autoclavable nanofibrous laminated composite fabrics with excellent waterproof and breathability were fabricated by electrospinning and lamination technology. The TPU/PEI nanofibrous membranes were hydrophobically treated and then laminated with two fabrics to construct sandwich structure composite fabrics. The laminated fabrics showed robust hydrostatic pressure of 38 kPa, high antisynthetic blood penetration of level 4, high filtration efficiency of 99.7%, and outstanding water vapor transmission rate of 5.2 kg m–2 d–1. Furthermore, benefiting from the excellent high-temperature resistance, dimensional stability, and outstanding tensile strength of TPU/PEI nanofibrous membranes, the as-prepared laminated fabrics could be autoclaved 10 times without great degradation of liquid barrier and breathability properties. The successful preparations of autoclavable nanofibrous laminated fabrics shed light on the reusable medical protective clothing design and provide useful guidance to solve the critical issue of the protective materials shortage.