For point-of-care testing (POCT), a sandwich-typed immunoassay on poly(dimethylsiloxane) (PDMS) microfluidic chips was developed by employing two original technologies: a power-free microchip and laminar flow-assisted dendritic amplification (LFDA). Sequential injection of reagents of immunoassay was carried out by the power-free microchip. In addition, for the amplification of a fluorescent signal, LFDA exploited the interaction between FITC-labeled streptavidin and biotinylated anti-streptavidin on the interface of the two streams of laminar flow during the sequential injection of the reagents of the immunoassay. The microfluidic immunoassay was finished within 21 min with a sample volume of 500 nL. The calibration curve of a quantitative analysis satisfied the concentration range of the cut-off values of prostate-specific antigen (PSA) in serum. The limit of detection (LOD) reached 520 pg/mL (16 pM) of PSA in human female serum. We believe that this microfluidic immunoassay and device are promising for POCT.