With the implementation and expansion of international sulfur emission control areas, effectively promoted the marine low sulfur diesel fuel (MLSDF) used in marine diesel engines. In this study, a large low-speed, two-stroke, cross-head, common rail, electronic fuel injection marine diesel engine (B&W 6S35ME-B9) was used for the study. According to diesel engine’s propulsion characteristics, experiments were launched respectively at 25%, 50%, 75%, 100% load working conditions with marine low sulfur diesel fuel to analyze the fuel consumption, combustion characteristics and emissions (NOx, CO2 , CO, HC) characteristics. The results showed that: Marine diesel engine usually took fuel injection after top dead center to ensure their safety control NOx emission. From 25% to 75% load working condition, engine’s combustion timing gradually moved forward and the inflection points of pressure curve after top dead center also followed forward. While it is necessary to control pressure and reduce NOx emission by delaying fuel injection timing at 100% load. Engine’s in-cylinder pressure, temperature, and cumulative heat release were increased with load increasing. Engine’s CO2 and HC emissions were significantly reduced from 25% to 75% load, while they were increased slightly at 100% load. Moreover, the fuel consumption rate had a similar variation and the lowest was only 178 g/kW·h at 75% load of the test engine with MLSDF. HC or CO emissions at four tests’ working conditions were below 1.23 g/kW·h and the maximum difference was 0.2 or 0.4 g/kW·h respectively, which meant that combustion efficiency of the test engine with MLSDF is good. Although the proportion of NOx in exhaust gas increased with engine’s load increasing, but NOx emissions were always between 12.5 and 13.0 g/kW·h, which was less than 14.4 g/kW·h. Thus, the test engine had good emissions performance with MLSDF, which could meet current emission requirements of the International Maritime Organization.