A dual parameter LSPR (localized surface plasmon resonance) biosensor using a single optical fiber for the simultaneous detection of two RNA targets has been proposed in this paper. The sensor co-immobilizes two types of Aunor’s (gold nanorods) with different AR (aspect ratio) on the same optical fiber and connects two types of sulfhydryl-modified ASOs (antisense oligonucleotides) via Au-S bonding to achieve two-parameter detection. The target binding induced sequence-specific hybridization between the RNA-airs in solution and the immobilized ASO-AuNRs complexes, resulting in a particle coupling effect. Experimental results confirmed the sensor’s effective detection of both single and dual parameters, with effective responses ranging from 0.001 to 50 nM. The sensor successfully recognized both N and E gene fragments within 15 min, while maintaining high selectivity and reproducibility. This integrated design addresses the limitations of traditional multiplexed LSPR detections and shows great potential for clinical diagnostics involving complex biological matrices.