Spent frying oil of fish was converted into biodiesel via alkali-catalyzed transesterification. Methyl and ethyl esters of this oil were successfully prepared and evaluated. The influence of transesterification parameters, such as KOH concentration, catalyst type (KOH and NaOH), methanol to oil, and the alcohol type, were investigated. Furthermore, a two-step transesterification of spent frying oil of fish was also conducted using KOH and NaOH with methanol. The results disclosed that 0.50% of KOH w/w of oil, a 6:1 methanol to oil, a reaction temperature of 60 degrees C, and a duration of 1 h were optimal conditions, for they resulted in best results. Thin layer chromatography of the optimum conditions sample was conducted on a silica gel plate. The fuel properties of the produced biodiesel were investigated according to ASTM standards. The results indicated that properties of spent frying oil of fish have been markedly enhanced by transesterification as compared to the direct use of it as a fuel. Blending of the parent oil and/or the optimum transesterified sample with petroleum diesel was made using different volume percentages. The results indicated that biodiesel had slight effects on the properties of petroleum diesel indicating a homogenized operative system.