Inefficiency, high cost, and complex operation have emerged as shackles for large-scale separate oil-in-water emulsion. Herein, a low-cost and eco-friendly separation layer with a rough structure and rich anionic groups was fabricated from rice straw (RS) via a simple acid-base treatment and slight squeeze process. The separation layer’s morphology, composition, and wettability were investigated. It was then employed to separate oil-in-water emulsion. The RS after acid and alkali treatment (A1A2-RS) exhibited a clear fiber structure and abundant humps, which made the separation layer superwettable and highly electronegative (−26.55 mV). The overlapped and intertwined A1A2-RS layer structure owned a superior performance for hexadecyl-trimethyl-ammonium-bromide (CTAB) adsorption and tiny oil interception. As a result, the separation layer had stable fluxes (>500 LMH) for multiple CTAB-stabilized emulsions and the obtained filtrates performed low total organic carbon (TOC) contents (<30 mg/L). In addition, the A1A2-RS layer had excellent renewability (10 cycles/ 200 mL) and the flux could be substantially recovered merely by aqueous wash. Moreover, filtrate analysis showed that the A1A2-RS layer had a good effect on actual emulsion treatment with a TOC removal rate of 89.56%.