In this study, we demonstrated an approach for the development of multiplex immunochromatographic test strip (MICS) for neomycin (NEO), penicillin (PEN), and chloramphenicol (CAP) detection using three different-colored gold nanoparticles. The gold nanoparticles: gold nanospheres (red), gold nanostars (blue), and gold nanoflowers (black) were applied as labels for MICS fabrication. The proposed MICS achieved a clearly visible limit of detection with cut-off values of 500, 5, and 0.5 μg L−1 for NEO, PEN, and CAP, respectively within only 10 min. Such smartphone-based detection provided a limit of detection of 3.70 μg L−1 for NEO, 0.10 μg L−1 for PEN, and 0.008 μg L−1 for CAP. The analysis of real samples by the developed MICS was well correlated with the enzyme-linked immunosorbent assay and liquid chromatography–mass spectrometry. The proposed MICS has the potential to be used for the simultaneous detection of NEO, PEN, and CAP with rapid, precise, and sensitive results.