Soil, as the largest terrestrial carbon sink, plays a crucial role in carbon sequestration. Within soil systems, microorganisms decompose soil organic matter to generate energy and obtain carbon for growth, concomitantly release heat and CO₂ as metabolic byproducts. The calorespirometric (CR) ratio – defined as the ratio of heat production to CO₂ evolution, is a key indicator of carbon use efficiency and soil anaerobicity. However, conventional methodologies typically measure heat and CO₂ separately, with CO₂ often quantified by intermittent sampling. This discontinuous approach, compounded by the inherent heterogeneity of soil, introduces uncertainties in calorespirometric analysis. To address this limitation, an infrared CO₂ sensor was mounted onto a stainless-steel calorimetric ampoule, containing soil-glucose mixtures, enabling simultaneous real-time measurements within an isothermal microcalorimeter. The novel configuration permits continuous monitoring of both parameters, validated through comparative analysis with traditional methods. The derived CR ratio aligned with theoretical predictions for carbohydrates metabolism. Furthermore, parallel oxygen measurements enabled quantification of CR ratio based on O₂ (heat-to-O₂), and the respiratory quotient (CO₂-to-O₂), offering deeper insight into microbial carbon-energy coupling and turnover in soil systems. This methodological advancement enhances the capacity to interrogate soil biogeochemical processes under dynamic environmental conditions.