Sugarcane mono-sequencing is one of the prominent systems of Uttar Pradesh but wide variations in cane yields are the main cause of concern across the cane producing zones. Hence, 131, 198 and 122 composite soil samples (0-20 cm depth) were taken from cane producing Eastern zone (EZ), Central zone (CZ) and Western zone (WZ) of Uttar Pradesh, respectively to assess the causes of yield variations and impact of sugarcane mono-sequencing on nutrient index (NI), microbial activity and soil quality. Sugarcane mono-sequencing depleted soil organic carbon (SOC), available nitrogen (Na), potassium (Ka), sulphur (Sa) and Zn as 48.9, 98.2, 39.0, 43.3 and 26.4% soil samples falls in low categories, respectively. However, soil degradation aggregated more in EZ soils due to acute deficiency of macro-nutrients as well as high content of Cu, Fe and Mn in low-lying areas. Total microbial counts, microbial biomass of C and N, basal soil respiration, acid and alkaline phosphatase were highest in WZ, but Na, Pa, Ka, Sa, FDA, β-glucosidase and urease activity was greater in CZ soils. As a result, NI was greater in CZ (2.19) followed EZ (1.98) and WZ (1.97), but soil quality index (SQI) was found in the order of CZ (0.795) >WZ (0.785) > EZ (0.708). The sensitivity index (S) was lowest in CZ soils (1.420) indicated that WZ (1.810) and EZ (1.915) soils are more preferable and sensitive to perturbations and management practices. Na was the most vital indicator for sugarcane production followed by SOC, AZO and Sa.