Molecular analysis has been widely used for disease diagnosis and treatment efficacy analysis. Plasmonic biosensors are popular for molecular analysis because of their rapid detection and high sensitivity. This paper presents a nanoplasmonic biosensor combined with a chemifluorescence approach for multiplex molecular profiling in plasma samples. The nanoplasmonic biosensor significantly improves the detection sensitivity with the fluorescence signal being enhanced by ~300-fold. In the measurement of multiple cytokines’ expression levels in patient plasma, the experimental results show that the nanoplasmonic biosensor achieves a limit of detection as low as 0.5 pg/mL. The assay time is as fast as 1 hour. We envisage that the developed nanoplasmonic biosensor has broad applications in disease diagnosis, including inflammation, cancer, or even infectious diseases.