The volume factor of crude oil is an important parameter for the understanding and evaluation of oil reservoirs in the process of exploration and development. Most of the evaluation unit information is missing due to the limitations of the high-pressure physical property(PVT) analysis of crude oil with direct access to crude oil volume factor data, such as sampling costs and conditions. The accuracy of the volume factor of crude oil determined by the currently most commonly used analogy or empirical formula method is significantly lowered. In order to solve this problem, the theoretical equation of crude oil volume factor is derived and established based on the definition of crude oil volume factor. In practical application, the newly established theoretical equation of crude oil volume factor is obviously superior to the one given in the definition of volume factor. The parameters involved in the new equation can be obtained by sampling analysis under ground conditions. The crude oil volume factor calculated by the new equation not only significantly reduces the cost of crude oil PVT sampling analysis, but also increases the calculation accuracy from 89.4% in the empirical formula to 96.5% in the new equation. The application range can be widened to any stage of oil reservoir exploration and development, and embraces great value for promotion.