<p>Molecular diagnostics based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) relies on temperature-dependent enzymatic reactions that require compatibility with thermal cycling processes. In this study, thermally responsive single-compartment polysaccharide microreactors were fabricated using a water-in-oil emulsion strategy combined with in situ agarose gelation. The effects of oil phase composition and agarose concentration on microsphere formation, size distribution, and protein encapsulation were systematically investigated, and isooctanol was identified as an effective oil phase for achieving stable emulsification with reduced interfacial leakage. Reverse transcriptase and DNA polymerase were subsequently encapsulated to construct single-compartment microreactors, and the biochemical compatibility of these microreactors was evaluated using a one-step RT-qPCR assay. The encapsulated enzyme systems exhibited amplification curves and cycle threshold values comparable to those of free enzyme controls, without detectable inhibition or kinetic interference. Moreover, multiplex RT-qPCR assays confirmed preserved detection specificity without apparent cross-reactivity. These results demonstrate that thermally responsive agarose microreactors provide a structurally stable and functionally neutral platform that is compatible with RT-qPCR workflows for molecular diagnostic applications.</p>