Expanding of karst rocky desertification (RD) area in southwestern China is strangling the sustainable development of local agricultural economy. It is important to evaluate the soil fertility at RD regions for the sustainable management of karst lands. The changes in 19 different soil fertility-related variables along a gradient of karst rocky desertification were investigated in five different counties belonging to the central Hunan province in China. We used principal component analysis method to calculate the soil data matrix and obtained a standardized integrate soil fertility (ISF) indicator to reflect RD grades. The results showed that the succession of RD had different impacts on soil fertility indicators. The changing trend of total organic carbon (TOC), total nitrogen (TN), available phosphorus, microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN) was potential RD (PRD) > light RD (LRD) > moderate RD (MRD) > intensive RD (IRD), whereas the changing trend of other indicators was not entirely consistent with the succession of RD. The degradation trend of ISF was basically parallel to the aggravation of RD, and the strength of ISF mean values were in the order of PRD > LRD > MRD > IRD. The TOC, MBC, and MBN could be regarded as the key indicators to evaluate the soil fertility.