The organohalide-respiring Sulfurospirillum multivorans uses chlorinated ethenes as electron acceptors for growth under anoxic conditions. However, little is known about the interaction of these substrates with proteins. Here, we apply thermal proteome profiling (TPP) to analyze enzyme-trichloroethene interactions. TPP is commonly used to investigate protein-ligand binding through protein melting curve shifts. Several modifications in the protocol, e.g. performing the incubation under anaerobic conditions and increasing the temperature range up to 97°C, improved the detection range and allowed the investigation of oxygen-sensitive proteins. Enzymatic reductive dehalogenation was prevented by omitting the electron donor during incubations. This enabled detecting the interaction of the tetrachloroethene reductive dehalogenase PceA with trichloroethene and confirms the enzyme’s specificity for this substrate. Another 19 proteins showed significant melting curve shifts with trichloroethene, pointing to other proteins directly or indirectly interacting with trichloroethene. Interestingly, a putative response regulator reacted similarly towards trichloroethene, which is potentially in line with its proposed role in regulating trichloroethene respiration. The TPP approach is here proven to facilitate the identification of substrate-enzyme interactions of strictly anaerobic reductive dehalogenases and probably their regulators. This strategy can be used to identify yet unknown substrate specificities and potential signal-sensing proteins in other difficult to study bacteria.