In this article, we report the results of the extent of interaction as well as the formation of a bioconjugate of human hemoglobin (lib) with silver (Ag). The complexation process and conformational changes are characterized using different spectroscopic and microscopic techniques. The UV-vis study demonstrates the perturbation of the soret/heme band and generates conformational heterogeneity within the heme group in the presence of silver. A fluorescence study suggests that the Tryptophan (Trp) residues of Hb are in a more polar environment after conjugation. Initial fluorescence enhancement with addition of silver is due to metal-enhanced fluorescence. Moreover, the fluorescence quenching after the formation of the Hb-Ag bioconjugate follows the modified Stern-Volmer (S-V) plot. The S-V plot along with the time-resolved fluorescence study indicates the presence of both static and dynamic types of quenching. In addition, the reduction potential values of the entities (lib heme, Ag+, and Trp) indicate the possible electron transfer. The secondary structure calculation from CD and FTIR spectra indicate a-helix to beta-sheet conversion, and unfolding of Hb is also responsible for the bioconjugate formation. In addition, FE-SEM, phase contrast inverted microscopy (PCIM) images demonstrate the formation of the silver protein bioconjugate. The overall data show that there is a change in the secondary as well as the tertiary structure of Hb after conjugation with silver.