The development of membrane technology is rapidly increasing due to its numerous advantages, including its ease of use, chemical resistant properties, reduced energy consumption, and limited need for chemical additives. Polyurethane membranes (PUM) are a particular type of membrane filter, synthesized using natural organic materials containing hydroxy (-OH) groups, which can be used for water filtration, e.g., ammonia removal. Red seaweed (Rhodophyta) has specific molecules which could be used for PUM. This study aimed to ascertain PUM synthesis from red seaweed biomass (PUM-RSB) by using toluene diisocyanate via the phase inversion method. Red seaweed biomass with a particle size of 777.3 nm was used as starting material containing abundant hydroxy groups visible in the FTIR spectrum. The PUM-RSB produced was elastic, dry, and sturdy. Thermal analysis of the membrane showed that the initial high degradation temperature was 290.71 °C, while the residue from the thermogravimetric analysis (TGA) analysis was 4.88%. The PUM-RSB section indicates the presence of cavities on the inside. The mechanical properties of the PUM-RSB have a stress value of 53.43 MPa and a nominal strain of 2.85%. In order to optimize the PUM-RSB synthesis, a Box–Behnken design of Response Surface Methodology was conducted and showed the value of RSB 0.176 g, TDI 3.000 g, and glycerin 0.200 g, resulting from the theoretical and experimental rejection factor, i.e., 31.3% and 23.9%, respectively.