Glutaraldehyde (GA) is a widely used cross-linking agent in biological research due to its superior characteristics, such as high reactivity toward proteins, high stability, and cost-effectiveness. In this regard, analyzing spectral changes initiated by various molecular forms and transformations of GA in a reaction medium and its reaction with surface functional-modified solid spheres is vital for a successful bioconjugation process targeting the biomolecules of interest. In this work, we present Fourier transform-infrared (FT-IR), Raman, and UV-visible spectroscopic analyses of glutaraldehyde-modified Fe3O4 microparticles (magnetic beads) to confirm the conjugation between GA and magnetic beads. We also studied the molecular transformations of glutaraldehyde during the reaction with amine-modified magnetic beads via investigating the reaction medium of the glutaraldehyde solution. Our FT-IR and Raman studies confirmed that glutaraldehyde was successfully coupled on the magnetic beads. Furthermore, FT-IR and UV-vis studies on the glutaraldehyde solution revealed the multiple molecular forms of GA in an aqueous medium, and they also confirmed that glutaraldehyde transforms into other molecular forms while the reaction occurs with the magnetic beads.