Hybrid nanoparticles composed of polydiacetylene (PDA) and magnetite (Fe3O4) were fabricated by a double-emulsion method. The structure and composite form of the hybrid nanoparticles were investigated with transmission electron microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy, and dynamic light scattering. The successful incorporation of magnetism to an attractive class of colorimetric PDA sensors was demonstrated. Compared with the PDA vesicles, the hybrid nanoparticles showed better colloidal stability with ethanol stimuli. In this study, we explored the colorimetric sensing ability of the hybrid nanoparticles in response to sodium cetyltrimethylammonium bromide (CTAB). The results show a high sensitivity to CTAB with a limit of detection at 30 μM. The biotin–streptavidin interaction was used as a sensing model to test ligand–receptor interactions on the hybrid nanoparticles' response. The concentration dependence of the chromic response was observed with the limit of visual detection at 20 μg/mL. The Fe3O4–PDA hybrid nanoparticles might have great potential applications in biosensing because of their advantages, including separation and sensing. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40634.