Colistin is recognized as the last therapeutic option for multidrug-resistant Gram-negative bacteria infection. In addition, bacterial resistance to colistin could be transmitted between different species through plasmid-mediated mcr-1 gene transfer. Therefore, rapid screening of colistin-resistant isolates will play a key role in controlling the spread of resistance and improving patient outcomes. We developed a rapid method for the detection of colistin-resistance in Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa bacteria based on Raman spectroscopy and hierarchical cluster analysis. Bacteria were incubated with and without colistin using CAMHB as the liquid culture medium. They were then centrifuged and dried on a glass slide. Five Raman spectra of each of the samples were recorded and analyzed by the hierarchical cluster analysis method to determine whether the bacteria were resistant. To evaluate this method, 123 clinical bacterial isolates (42 isolates of E. coli, 41 isolates of A. baumannii and 40 isolates of P. aeruginosa) were tested. The detection sensitivity and specificity were 90.9% and 91.1%, respectively, compared with the reference broth microdilution method. The screening is easy to perform and can be completed in 1.5 h, suggesting that it holds great potential to be an initial screening method in countries and areas where colistin becomes the last resort antibiotic.