The study of the thermodynamic properties of engine fuels and the in-cylinder gas is involved in the analysis of chemical compound reaction and the thermodynamic analysis of fuel and gas, which is quite important in engine combustion investigations because the fuel chemical energy converts into internal work energy during this stage. Although the research on the thermodynamic properties of fuel and in-cylinder gas has lasted a few decades, with the development of new fuel types, developing general models with sufficient accuracy to calculate the fuel and in-cylinder gas thermodynamic properties for engines remains a challenge. This paper presents a model to calculate the diesel fuel and in-cylinder gas thermodynamic properties based on the mixture composition theory and considering the diesel fuel and in-cylinder gas mixtures in terms of the chemical reaction fundamentals. The diesel fuel and in-cylinder gas thermodynamic property modeling approach for the combustion investigation is then applied in the heat-release calculation model of a marine diesel engine, which is validated by experimental research on heat release. According to the simulation and experimental results, when considering that the diesel fuel and in-cylinder gas thermodynamic properties are affected by the in-cylinder temperature, fuel type, and air excess ratio, the engine combustion simulation results in predictions that are more accurate than those when setting the values constant. This paper provides a general approach for the investigation and application of engine fuels and in-cylinder gas thermodynamic properties, in particular for new fuel substitution in engines.