Vitamin C (VC) and Vitamin B9 (VB9) are essential micronutrients integral to numerous biological functions and critical for maintaining human health. The rapid detection of these vitamins is important for verifying nutritional supplements and aiding in clinical diagnoses. This study combined terahertz time-domain spectroscopy (THz-TDS) with metasurface technology to develop a fast, sensitive, and non-destructive detection method for VC and VB9. Firstly, we determined the characteristic absorption peaks and molecular vibration modes of VC and VB9 within the 0.5–4.0 THz range through quantum chemical calculation and THz-TDS measurement. Then, we designed and fabricated a metasurface biosensor to match its resonance peak with the communal peak of VC and VB9, enhancing the interaction between THz waves and these vitamins. Using this biosensor, we analyzed solutions with different concentrations of VC and VB9. An increase in vitamin concentrations resulted in frequency shifts in the THz resonance peak. Quantifiable relationships between frequency shifts and the vitamin concentrations were established. The detection limits achieved were 158.82 ng/µL for VC and 353.57 ng/µL for VB9, respectively. This method not only demonstrates high sensitivity but also simplifies the operational process, offering an innovative tool for applications in food safety monitoring and clinical diagnostics.