A new unknown impurity of isosulfanblue formed during the synthesis of isosulfanblue, and the level of the impurity was found at below 0.10%. This impurity was identified by Liquid Chromatography-Mass Spectrometry (LC-MS) and characterized by Proton Nuclear Magnetic Resonance (1H-NMR), Carbon-13 Nuclear Magnetic Resonance (13C-NMR), MS and Fourier-Transform Infrared Spectroscopy (FTIR). Based on the spectral data, the impurity was named as, N-4-[bis[4-(diethyl amino)phenyl] hydroxymethyl]-benzene-2,5-disulphonic acid. This impurity was isolated by preparative High-performance Liquid Chromatography (HPLC) and co-injected into HPLC to confirm the retention time. Isolation, structural elucidation of the impurity and its possible mechanism of formation has been discussed. To the best our knowledge, the formation, identification, isolation, characterization of the observed new impurity was not mentioned in the literature till date.