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A novel wavelength modulated optical fiber-based symmetrical long-range surface plasmon resonance (sLRSPR) biosensor with high quality factor (Q-F) of 56.70 RIU−1 and temperature self-reference was proposed and demonstrated. The multilayered architecture of the sLRSPR biosensor consisted of cladding/dielectric-buffer-layer (DBL)/Au-nanofilm/DBL/analyte, and the DBLs on both sides of the Au-nanofilm were identical, which greatly increased the matching degree of the permittivities of analyte and the DBL near cladding and reduced the loss of long-range surface plasmon-polaritons. Therefore, the sLRSPR sensor displayed excellent sensing performance, and its Q-F was 3.27-folds higher than that of the conventional SPR sensor. Furthermore, the sLRSPR sensor was employed as a biosensor for the measurement of average refractive index (RI) of cell, and the temperature-sensitive optical adsorption characteristic of the DBL was utilized to provide temperature self-reference for the RI measurement. The sLRSPR biosensor realized simultaneous measurement of RI and temperature at the exactly same location, which can eliminate the measurement error introduced by temperature to RI measurement. The sLRSPR biosensor proposed in this paper possessed higher detection precision and presented greatly promising application prospects in the field of biochemistry.