Natural resources and environmental audit is an important system for the in-depth implementation of the strategy of ecological civilization construction, and it serves as a crucial means to ensure the coordinated development of China’s green and low-carbon transition and economic and social high-quality development. The necessity of developing the disciplinary direction of natural resources and environmental audit primarily includes national strategic requirements, industry demands, and academic needs; its feasibility can be manifested by the formation of an independent professional field, proactive actions by major universities, and substantial progress achieved by Nanjing Audit University. The construction of the disciplinary direction in natural resources and environmental audit should adhere to the basic principles of the “Five Persistences”, and build the overall “3+X” framework of natural resources and environmental audit encompassing national natural resources and ecological environment audit, internal corporate natural resources and environmental audit, social natural resources and environmental audit, environmental engineering audit, intelligent natural resources and environmental audit, and international natural resources and environmental audit. Finally, several measures are proposed to accelerate the development of the disciplinary direction in resources and environmental audit, including enhancing the strength of the academic team, solidifying the professional foundation of the discipline, reinforcing cooperation with the practical sector, and seizing opportunities related to ESG audit and carbon audit.