Polysiloxane-based polyurethane/lignin elastomers are synthesized via pre-polymerization. Isophorone diisocyanate (IPDI) reacts with polycarbonate (PCDL) and polydimethylsiloxane (PDMS) polyol to produce a linear thermoplastic elastomer. Lignin is added to the matrix of the polyurethane, where lignin not only acts as a crosslinking agent but also as filler. The effects of polysiloxane-based polyurethane/lignin elastomers on thermal properties, mechanical properties, surface hydrophobicity, and cytotoxicity in vitro are investigated. The heat resistance, surface hydrophobicity and mechanical strength of the materials are improved, and a decreased cytotoxic behavior against HeLa cells by MTT assay is observed, which should be attributed to the semi-interpenetrating network structure and low toxicity of lignin, respectively. With the addition of 1 wt% of lignin, cell viability increases to 65.2%, mechanical strength increases to 17.1 Mpa and contact angle increases to 108.2°.