Copper-thiolate self-assembly nanostructures are a unique class of nanomaterials because of their interesting properties such as hierarchical structures, luminescence, and large nonlinear optical efficiency. Herein, we synthesized biomolecule cysteine (Cys) and glutathione (GSH) capped sub-100 nm self-assembly nanoparticles (Cu-Cys-GSH NPs) with red fluorescence. The as-synthesized NPs show high emission enhancement in the presence of ethanol, caused by the aggregation-induced emission. We correlated the structure and optical properties of Cu-Cys-GSH NPs by measuring the mass, morphology, and surface charge as well as their two-photon excited fluorescence cross-section (σ2PEPL), two-photon absorption cross-section (σTPA) and first hyperpolarizability (β) of Cu-Cys-GSH NPs in water and water-ethanol using near-infrared wavelength. We found a high β value as (77 ± 10) × 10-28 esu (in water) compared to the reference medium water. The estimated values of σ2PEPL and σTPA are found to be (13 ± 2) GM and (1.4 ± 0.2) × 104 GM, respectively. We hope our investigations of linear and nonlinear optical properties of copper-thiolate self-assemblies in water and its solvent-induced aggregates will open up new possibilities in designing self-assembled systems for many applications including sensing, drug delivery, and catalysis.