The fragile eco-environment, together with low efficiency energy utilization, are typical features of the social-ecological system in Karst mountain areas. It is of great value to determine the potential agricultural biomass resources (ABR) for rural energy construction, and ecological environmental protection of these areas. Based on a case study of Guizhou Province, considering the returning ecological straw biomass for soil and water conservation, this paper estimated the total available ABR in rural areas using the residue to product ratio, ecological returning coefficient, collection coefficient, conversion coefficient to standard energy, and process residue factor. The results showed that:(1) from 2009 to 2015, the ABR of Guizhou Province increased to 5191.50×10⁴ tce annually, with a density of 295 tce/km² and 1.48 tce/person, respectively. About 1974.05×10⁴ tce of energy-oriented biomass resources are available annually, accounting for 21.85% of the total energy consumption during the same period in Guizhou; (2) the proportion of biomass energy reserves in Guizhou Province is unevenly distributed. The potential utilization of livestock and poultry waste is considerable, with a contribution value up to 85%; the average amount of cattle feces biomass is up to 3009.78×10⁴ tce annually, with a 68.39% contribution to the total animal excrement, which is the most important ABR in Guizhou Province; (3) a U-shaped temporal characteristic change in ABR occurred during 2009 to 2015 in Guizhou Province, with the lowest level in 2011, which showed a coupling with an important ecological event of the southwest drought disaster in 2010. Finally, based on the current agricultural biomass resources in Guizhou Province, this paper suggests that the straw power plants and straw curing for fuel factories should be distributed evenly in the karst mountain area, improving the current scale of livestock breeding, strengthening agricultural disaster prevention, and popularizing the comprehensive utilization of biogas, efficient firewood stoves, and energy saving straw gases in karst rocky desertification areas.