Rapid and sensitive methods to detect proteins and protein denaturation have become increasingly needful in the field of proteomics, medical diagnostics, and biology. In this paper, we have reported the synthesis of a new cationic water-soluble conjugated polymer that contains fluorene and diene moieties in the backbone (PFDE) for protein identification by sensing an array of PFDE solutions in different ionic strengths using the linear discriminant analysis technique (LDA). The PFDE can form complexes with proteins by electrostatic and/or hydrophobic interactions and exhibits different fluorescence response. Three main factors contribute to the fluorescence response of PFDE, namely, the net charge density on the protein surface, the hydrophobic nature of the protein, and the metalloprotein characteristics. The denaturation of proteins can also be detected using PFDE as a fluorescent probe. The interactions between PFDE and proteins were also studied by dynamic light scattering (DLS) and isothermal titration microcalorimetry (ITC) techniques. In contrast to other methods based on conjugated polymers, the synthesis of a series of quencher or dye-labeled acceptors or protein substrates has been avoided in our method, which significantly reduces the cost and the synthetic complexity. Our method provides promising applications on protein identification and denaturation detection in a simple, fast, and label-free manner based on non-specific interaction-induced perturbation of PFDE fluorescence response.