Recently, the coupling of biofuel fermentation broths and pervaporation has been receiving increasing attention. Some challenges, such as the destructive effects of constituents of the real fermentation broth on the membrane performances, the lethal effects of the membrane surface chemical modifiers on the microorganisms, and being expensive, are against this concept. For the first time, a continuous study on the one-step and low-cost preparation of superhydrophobic membranes for bioethanol separation is made to address these challenges. In our previous work, spraying as a fast, scalable, and low-cost procedure was applied to fabricate the one-layered active-layer hydrophobic (OALH) silicalite-1/polydimethylsiloxane (PDMS) membrane on the low-cost mullite support. In this work, the spraying method was adopted to fabricate a two-layered active-layer superhydrophobic (TALS) silicalite-1/PDMS membrane, where the novel active layer consisted of two layers with different hydrophobicities and densities. Contact-angle measurements, surface charge determination, scanning electron microscopy, atomic force microscopy, and pervaporation separation using a 5 wt % ethanol solution were used to statically evaluate the fouling/biofouling resistance and pervaporation performances of OALH and TALS membranes in this study. The TALS membrane presented a better resistance and performance. For dynamic experiments, the Box− Behnken design was used to identify the effects of substrates, microorganisms, and nutrient contents as the leading indicators of fermentation broth on the TALS membrane performances for the long-term utilization. The maximum performances of 1.88 kg/m 2 • h, 32.34, and 59.04 kg/m 2 •h concerning the permeation flux, separation factor, and pervaporation separation index were obtained, respectively. The dynamic fouling/biofouling resistance of the TALS membrane was also characterized using energy-dispersive X-ray spectroscopy of all the tested membranes. The TALS membrane demonstrated the synergistic resistance of membrane fouling and biofouling. Eventually, the novel TALS membrane was found to have potential for biofuel recovery, especially bioethanol.