Heme oxygenase-2 (HMOX-2) has recently emerged as a critical regulator of tumor progression and metastasis. Tumor-initiating cell near-infrared probe (TiNIR) has been identified as a promising small molecule with inhibitory activity against HMOX-2, yet the molecular basis of its subtype selectivity remains unclear. This study sought to elucidate the structural and mechanistic foundations of TiNIR’s preferential binding to HMOX-2 over HMOX-1 and to assess its potential as a theranostic agent in metastatic lung cancer. A combined experimental and computational strategy was employed. Recombinant protein binding assays and lung cancer cell models (PC-9, A549) were used to validate TiNIR’s selective interaction with HMOX-2. Structure-based docking, pharmacophore modeling, and molecular dynamics simulations provided mechanistic insights into binding stability and selectivity. TiNIR displayed markedly higher affinity for HMOX-2 than HMOX-1 at both protein and cellular levels, with fluorescence intensity correlating to HMOX-2 expression. Docking analyses revealed that TiNIR occupied the heme-binding domain of HMOX-2 with binding energies comparable to or exceeding those of heme, while steric and electrostatic constraints limited binding in HMOX-1. Pharmacophore modeling confirmed that the open and accessible pocket of HMOX-2 enabled stable multipoint interactions, whereas HMOX-1’s narrower cavity restricted TiNIR accommodation. Molecular dynamics simulations further demonstrated long-term conformational stability of the TiNIR–HMOX-2 complex through consistent non-covalent interactions. These findings establish TiNIR as a structurally validated, HMOX-2-selective small molecule with both diagnostic and therapeutic potential in metastatic cancer. By competitively occupying the heme-binding site, TiNIR effectively inhibits HMOX-2 activity, offering a new molecular framework for the development of subtype-specific anticancer strategies.