Ex vivo engineering of organs that uses decellularized whole organs as a scaffold with autologous stem cells is a potential alternative to traditional transplantation. However, one of the main challenges in this approach is preparing cytocompatible scaffolds. So far, high-precision and specific evaluation methods have not been developed for this purpose. Cell-based biosensors (CBBs) are promising tools to measure analytes with high sensitivity and specificity in a cost-effective and noninvasive manner. In this paper, using the NF-κB inducible promoter we developed a CBB for residual detergent detection. Proximal and core sections of the inducible promoter, containing NF-κB binding sequence, are designed and cloned upstream of the reporter gene (secreted alkaline phosphatase (SEAP)). After transfection into HEK293 cells, stable and reliable clones were selected. After confirmation of induction of this gene construct by sodium dodecyl sulfate (SDS), the stability and function of cells treated by qPCR and SEAP activity were measured. This biosensor was also used to evaluate the cytocompatibility of decellularized tissue. Results showed that the developed biosensor could detect very small amounts of SDS detergent (3.467 pM). It has the best performance 8 h after exposure to detergent, and its stability in high passage numbers was not significantly reduced. Applying this biosensor on decellularized tissues showed that SEAP activity higher than 4.36 (U/L) would lead to a viability reduction of transplanted cells below 70%. This paper presents a novel method to evaluate the cytocompatibility of decellularized tissues. The developed CBB can detect residual detergents (such as SDS) in tissues with high sensitivity and efficiency.