In this work, we developed a fast and straightforward colorimetric and photoluminescent chemosensor probe (P1), featuring bis-thiophene-thiosemicarbazide moieties as its signaling and binding unit. This probe exhibited rapid sensitivity to Hg2+ and Cu2+ ions in a semi-aqueous medium, resulting in distinct colorimetric and photoluminescent changes. In the presence of Cu2+, P1 displayed an impressive 50-fold increase in photoluminescence (PL) at 450 nm (with excitation at 365 nm). The probe P1 formed a 1:1 complex with Hg2+ and Cu2+ ions, featuring association constant values of 4.04 × 104 M−1 and 1.25 × 103 M−1, respectively. P1 has demonstrated its efficacy in the analysis of real samples, yielding promising results. Additionally, the probe successfully visualized copper ions on a mouse fibroblast cell line (NIH3T3), highlighting its potential as an intracellular probe for copper ion detection.