Abstract: Farmers cooperatives have been playing more and more important roles in agricultural and rural economic development in underdeveloped regions. However, a variety of alienation phenomena emerged in the course of its development and hindered the healthy growth and sustainable development of farmers cooperatives. Based on the Rochdele principle, the international cooperative principle, and the cooperative law of the People's Republic of China, a comprehensive measurement index system for the alienation of farmers cooperatives was constructed and used to measure the comprehensive alienation of farmers cooperatives based on the survey data in Gansu Province. Analysis results showed that, in general, the tendency towards the alienation of farmers cooperatives in Gansu Province was high with a level of 40.12%. The surplus distribution has the highest level of alienation of 50.75%, the system construction and democratic management levels have reached 35.53% and 33.42% respectively, and the alienation value of the freedom of joining in and dropping out was relatively low, only 22.64%. The inefficient operation mechanism and imperfect institution were the main reasons causing the alienation of farmers cooperatives. Based on this research, it is suggested that some countermeasures including formulating the statutes, strengthening democratic management, enhancing the mechanism of surplus distribution, and improving related laws and regulations should be adopted to support the healthy and sustainable development of farmers cooperatives.