For protein analysis, the current peptide-based probes rely almost on the specific recognition of the protein while neglecting the potential influence of the environment near the protein. Herein, we propose that to achieve high recognition of transmembrane protein integrin αvβ3, the interactions from the membrane substrate could be helpful. Moreover, to guarantee the additive effect of different interactions, the cis and trans isomers of peptide-based probes are distinguished. In detail, we synthesized the peptide-conjugated cis/trans isomers (cis-RTP and trans-RTP) by modifying the Arg-Gly-Asp (RGD)-targeting peptide and palmitic acid-conjugated Arg-Arg-Arg-Arg (Pal-RRRR) peptide to the two ends of the molecular scaffold-tetraphenylethene derivative. Due to the difference in spatial structure, isothermal titration calorimetry and simulation experiments demonstrated that cis-RTP can bind more stably to integrin αvβ3 than trans-RTP. As a result, cis-RTP has shown more excellent properties in inhibiting cell migration and killing cells by regulating actin and extracellular signal-regulated kinase. Unlike the existing probe design for protein, this study provides a concept of microenvironment-helpful recognition and a promising strategy of cis/trans isomers to modulate the interaction between proteins and probes.