Carbon-based nanoparticles (CNPs) have emerged as novel man-made materials with diverse applications, which may present significant risks to organisms. To bridge the gap in our knowledge of nano-toxicology, a number of in vitro or in vivo studies have been carried out. However, toxicity data remains limited. Herein, we employed a biospectroscopy approach to assess CNP-induced effects in zebrafish (Danio rerio). Zebrafish were exposed to Fullerene (C60), long or short multi-walled carbon nanotubes (MWCNTs), or single-walled carbon nanotubes (SWCNTs) for 21 days at two concentrations: 0.1 mg L-1 or 0.001 mg L-1. Following exposure, the brain, gills, gonads and liver from zebrafish were interrogated by attenuated total reflection Fourier-transform infrared (ATR-FTIR) or Raman spectroscopy. Computational analysis was then applied to the acquired infrared (IR) spectra, and distinct biochemical segregations between the exposed tissues vs. control were observed with spectral biomarkers of alterations identified. In addition, lipid-to-protein ratios in all four tissues were calculated by the IR spectra; unsaturated lipid levels in brain and gonad were assessed by Raman spectroscopy. Marked lipid alterations were observed. These findings show that biospectroscopy approaches have the potential to detect CNP-induced biochemical alterations in zebrafish.