Fossil fuel energy crisis and environmental pollution have initiated the scientific research on alternative fuels. Biodiesel (B100), gas to liquid (G100), and coal to liquid (C100) are superb selections to be substitutes for conventional diesel. To better investigate the emission characteristics of the alternative fuels mentioned above, a portable emission measurement system (PEMS) was used to carry out this study under real-world driving conditions. Results showed that the driving conditions had a notable effect on the vehicle emissions, the CO, THC, and CO2 emissions were higher under urban condition, and the NOx, PM (particle mass), and PN (particle number) emissions were higher under suburban condition. The expressway condition resulted in lower emissions except for PN due to more nucleation particles emitted. The use of B100, G100, and C100 fuels led to a reduction of more than 50% in the CO emission, especially for the C100, but the reduction effects for the THC were not obvious, and among them, G100 is the most prominent. Higher NOx emission was emitted after using the three fuels, especially for the B100; meanwhile, B100 increased the CO2, but G100 and C100 decreased the CO2 emission compared with D100. The PN emissions reduced by 1-2 orders of magnitude in comparison with those from D100 after using the three alternative fuels, and more than 50% of the PM could be reduced. B100 has the most significant particle reduction effect due to its oxygen-containing property, and it produced an evidently higher proportion of nucleation particles than D100, followed by G100 and C100.