The invention discloses a cell signal transduction activity detection method based on a tetrahedral DNA nano-material, which comprises a specific DNA binding sequence of a transcription factor capable of characterizing signal transduction activity to be detected and a fluorescent label, when a target transcription factor is bound with TDNs, a fluorophore and a quenching group are prevented from approaching, and when the target transcription factor is bound with TDNs, the signal transduction activity to be detected is detected. Therefore, the transcription factor activity can be analyzed by detecting the fluorophore. The method is simple, convenient and efficient, qualitative, quantitative and imaging analysis can be synchronously completed, and the operation time is short. In addition, the synthesis method of the tetrahedral DNA nano material is simple and convenient, has good reproducibility, can be flexibly designed according to target molecules to be detected, and has strong universality.