This study presents a novel method for direct amplification of multiple microRNAs (miRNAs) from serum samples using Sensitive and Multiplexed One-Step RT-qPCR (SMOS-qPCR). The technique eliminates the need for separate miRNA extraction and purification steps, offering a streamlined approach for non-invasive early disease diagnosis. We optimized reaction conditions, including serum treatment methods and PCR system volumes, to enhance interference resistance and detection sensitivity. The optimized serum direct SMOS-qPCR demonstrated a detection limit as low as 6 × 10