Background: Circulating cell-free mRNA (cfmRNA) expression can be considered as a compendium of transcripts collected from all organs. It has the capability of integrating functional and genetic information of tissues, highlighting this analyte’s unique potential as a non-invasive biomarker in early detection, therapy selection and patient follow-up of cancer management. Plasma cfmRNA is usually made up of degraded small fragments of smaller than 200 nucleotides, very low concentration, and with different terminal modification, these properties make it difficult to detect. We have developed, validated and automated a cfmRNA clinical testing workflow for simultaneous measurement of PD-L1 expression, ALK, ROS1 and NTRK fusions. This proprietary real-time qPCR-based process is exosome-free, highly sensitive and requires only half milliliter of plasma with 72-hour turnaround. A plasma cfmRNA profiling database covering 750 genes in 9 major cancer pathways was also established with novel cancer type-specific characteristics. Methods: Circulating cfRNA was extracted from 400 uL of plasma and reverse transcribed to cDNA. The cDNA pool then served as the universal source for multi-biomarker tests. All target primer and probe sets were selected based on RNA secondary structures. Plasma PD-L1 expression, ALK, ROS1, NTRK fusions and transcriptomic profiling were performed by Circulogene CLIA/CAP-complied testing platform. Results: Limit of detection (LOD) for PD-L1, ALK/ROS1 and NTRK were 1.0 copy/uL, 17.5 copies/uL and 28 copies/uL, respectively. For scoring of PD-L1 expression, based on Keynote trials, we used the 30th percentile Ct value as cutoff in qPCR which corresponded to IHC ≥ 50% TPS; while the 66th percentile Ct value corresponded to IHC ≥ 1% TPS. PD-L1 cfmRNA was demonstrated to be an excellent surrogate marker of tissue PD-L1 protein for clinical outcomes with immunotherapy. In a global cfmRNA landscape, a functional transcriptomic databank was also established, including differential gene expression, classification, functional clustering and cancer type-specific signatures. Conclusions: Liquid biopsy qPCR tests targeting PD-L1 expression, ALK, ROS1 and NTRK fusions are commercially available and filling the gap of tissue-based assays and NGS. Our groundbreaking cfmRNA work has research, clinical, and diagnostic value, and provides greater dimensionality to the current knowledge of cfmRNA research and makes a significant jump into understanding and devising strategies to tailor cancer Dx and Rx.