A smartphone-assisted microchemistry analyzer with an image-based colorimetric assay using ImageJ and a smartphone app detection method was successfully developed for the quantitative detection of melamine (MEL). The color changes of the MEL in the colloidal gold lateral flow immunoassay strip were captured and analyzed using a smartphone-controlled analyzer with an LED light source and a smartphone camera. The smartphone camera and light source were used to read the colorimetric signal from the strip, and a smartphone app was written and installed onto the smartphone. The quantitative analysis was validated with ImageJ and smartphone app colorimetric analysis. The limits of detection (LODs) for ImageJ and the smartphone app were calculated as 0.30 and 0.07 mg/L, respectively. The highly quantitative relationships between the MEL concentrations and the optical density and gray value of the ImageJ and smartphone app detection method. The designed image-based biosensor is successfully applied to detect MEL in solution of standard MEL and commercial milk samples.