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In this study, Mo3Se3–single-chain atomic crystals (SCACs) with atomically small chain diameters of ∼0.6 nm, large surface areas, and mechanical flexibility were synthesized and investigated as an extracellular matrix (ECM)-mimicking scaffold material for tissue engineering applications. The proliferation of L-929 and MC3T3-E1 cell lines increased up to 268.4 ± 24.4% and 396.2 ± 8.1%, respectively, after 48 h of culturing with Mo3Se3–SCACs. More importantly, this extremely high proliferation was observed when the cells were treated with 200 μg mL–1of Mo3Se3–SCACs, which is above the cytotoxic concentration of most nanomaterials reported earlier. An ECM-mimicking scaffold film prepared by coating Mo3Se3–SCACs on a glass substrate enabled the cells to adhere to the surface in a highly stretched manner at the initial stage of cell adhesion. Most cells cultured on the ECM-mimicking scaffold film remained alive; in contrast, a substantial number of cells cultured on glass substrates without the Mo3Se3–SCAC coating did not survive. This work not only proves the exceptional biocompatible and bioactive characteristics of the Mo3Se3–SCACs but also suggests that, as an ECM-mimicking scaffold material, Mo3Se3–SCACs can overcome several critical limitations of most other nanomaterials.