Circularly polarized luminescence (CPL) is a fascinating luminescence phenomenon that has garnered significant research attention for chiroptical applications. In this study, we developed a highly sensitive chiroptical sensor by co-assembling G-quartet nanofibers and nonchiral nitrogen sulfur-doped carbon dots (N-S-CDs) for dual ion detection. The N-S-CDs were synthesized using the one-step microwave method, and a helical G-quartet-based nanofiber structure (g-fiber) was simultaneously formed from guanosine 5'-monophosphate (GMP) in the presence of Sr2+. An adjustable helical G-quartet-based nanofiber provided an optimal chiral environment for CPL emission, with a dissymmetry factor (glum) reaching ±0.02. Notably, the left-handed (L-) and right-handed (R-) helical chirality of the complex was determined by switching between kinetic trap states and thermodynamic equilibrium during the reaction process. An optimized CPL sensor was developed based on chiral CDs/g-fiber composite materials, utilizing sensitive CPL as the signal output of dual detection for Hg2+ and I-. Similar limits of detection (LODs) were achieved for both L-/R-nanocomposites, with the best results being 83.5 nM for Hg2+ and 142.8 nM for I-. These values are comparable with or even better than those obtained with other optical analytical methods. Since CPL biosensors are relatively rare to date, our work presents a new horizon for the application of chiral CPL composites in biological assays.