Biodiesel (methyl ester) was synthesized from cottonseed oil (CSO) with methanol in the presence of an alkali catalyst (KOH) through the transesterification method. Response surface methodology (RSM) optimized the methyl ester synthesis process. The experimental design matrix was arranged using the Box-Behnken Design (BBD) of RSM. The optimization of methyl ester was achieved by varying variables such as the methanol to CSO ratio, the concentration of the catalyst, and the reaction duration. A contour plot and a surface plot were generated to illustrate the impact of these variables on methyl ester synthesis. The biodiesel yield was modeled as a quadratic equation through RSM analysis, with a coefficient of determination (R2) and adjusted R2 of 0.97 and 0.968, respectively. The optimized CSOME yield (95.04%) was numerically gained at a 1:6 methanol to CSO ratio, a reaction duration of 52.2 min, and a 0.985 wt% catalyst concentration. Based on these parameters, a 94.80% CSOME yield was experimentally obtained. This shows that response surface methodology (RSM) is sufficient for optimizing methyl ester yield. The produced methyl ester was characterized to determine its physicochemical properties. Gas chromatography-mass spectrometry (GC-MS) analysis was used to identify the specific type and proportion of fatty acid esters present in the CSOME. The main types of fatty acids found in CSOME were methyl 9-cis 11-trans-octadecadienoate (C18:2), hexadecanoic acid methyl ester (C16:0), 11, 14-octadecadienoic acid methyl ester (C18:2), methyl 8, 9-octadecadienoate methyl ester (C18:2), and myristic acid methyl ester (C14:0).