Consuming traditional petroleum-derived diesel fuel has long been associated with issues such as the depletion of natural energy resources. To solve these challenges, an alternate source like as biodiesel is an appealing option. Seed oils have long been recognized as an abundant and diverse source of biodiesel. In this study, poppy seed oil from the poppy (Papaver somniferum) was investigated for biodiesel production. Poppy seed biodiesel was generated and refined using acid-pretreated esterification with sulphuric acid prior to transesterification, as well as single-step alkaline catalyzed transesterification with methanol and potassium hydroxide. Finally, the percentage yield was compared. Using Statistica, the Box-Behnken design was applied to optimize process variables like time, temperature, catalyst concentration, and methanol-oil ratio to produce maximum yield. The relationship of process variables was also shown with the help of the Response Surface Methodology. A maximum yield of 94.87 % was obtained at optimized conditions, i.e., 90min reaction time, 60 °C of temperature, 0.25 mg of catalyst concentration, and 3v/v% alcohol-oil ratio. The fuel properties of biodiesel produced, such as acid value, moisture content, saponification value, iodine value, specific gravity, percentage of free fatty acids, refractive index, viscosity, boiling point, and peroxide value, were measured and compared with the American Society for Testing and Materials (ASTM) D6751 and European Standards (EN) 14214. Further results were studied and discussed using Fourier Transfer Infrared (FTIR) analysis, which showed maximum similarity of raw material to formed biodiesel. Gas Chromatography-Mass Spectrometry (GC-MS) analysis was performed to identify and quantify various fatty acid methyl esters. The results obtained were in accordance with various international standards for biodiesel fuel. Thus, poppy seeds can be used to obtain biodiesel.