Due to the increasing price of crude oil, biofuel as an alternative of fossil fuels is sought to be a global-wide solution to reduce the emission of greenhouse gas. In order to compare the influence on engine which meet euro IV emission standard by using pure fossil diesel, 2% and 5% biodiesel. A long-term operation with blended 2% and 5% biodiesel by standard engine test had been evaluated in this study. The results could be referred to the consideration of marketing promotion strategy as well as government policy in Taiwan. Both B2-B5 diesel fuel (2 and 5 vol% biodiesel) and diesel fuel were employed to the same engines for testing purpose to compare the influence. The engines were equipped with an electronically controlled common-rail fuel injection system. After 500 hours full load durability operation, the key components of the engines were analyzed and characterized by a series of inspections to identify the degree of decay and wear behavior. In addition, the engine performance which by the test procedure for SAE J1995 and emission measured which by the test procedure for FTP transient cycle were also determined at each maintenance stage. In this study, oil characteristics after durability test had also been evaluated. The results indicated that the engine components including piston rings, cylinder liners and needle valves of injector were less difference in dimensions comparison before and after durability tests. The fuel supply system of the diesel engine consists of a fuel tank, fuel pipes, fuel filters, and a low pressure pump had also been evaluated. According to microscopic characterization and engine oil analyses, the wear loss of the key components was insignificant and tribological performance of the B5 biodiesel was slightly better than that of diesel fuel. It was found that the performance and emissions were varied under ±3% when diesel engine was fueled with B5 biodiesel.