Abstract: There is growing concern about the negative impact of massive fertilizer use on soil health, threatening the environmental quality and sustainability of global agricultural production. Microbial metabolic quotient (qCO2) is an important soil health indicator, used to measure microbial C use efficiency (CUE). Soil carbon sequestration can be enhanced by increasing soil microbial biomass and CUE, ultimately improving soil health. However, the effects of long-term fertilization of agricultural soils on qCO2 variability remain unclear. This review provides a deep insight into the concepts and factors that influence qCO2 stability. Soil qCO2 depends on nutrient availability, and long-term fertilization increases microbial CUE in terms of decreased qCO2 values. Therefore, it is necessary to identify several theoretical advances and practical implications of qCO2 for microbial control of soil carbon cycling. In addition, qCO2 can be used as a tool for ecosystem monitoring and assessment in remediation programs due to its low-cost measurement.