This study focuses on the creation and comprehensive optical characterization of agar-based phantoms with varying concentrations of Indian ink and Al2O3 nanoparticles. The phantoms were designed to simulate human tissue for biomedical applications. We measured the total transmittance (%T), total reflectance (%R), and collimated transmittance (%Tc) to calculate the absorption (μa) and scattering (μs) coefficients. An optimization algorithm was applied to minimize the error between theoretical and experimental values of reflectance and transmittance, refining the refractive index (noptimized). Results show that Indian ink concentration significantly affects μa (p = 0.0198) and noptimized (p < 0.001), while nanoparticle concentration influences μs (p < 0.001). These findings advance the precision of tissue phantoms for biomedical applications.