An apparatus for detecting macromolecules in a biological fluid is discloses which includes a first substrate and a second substrate. The first substrate includes a plurality of sampling apertures, at least some of which have different diameters. The sampling apertures are configured to screen and isolate the macromolecules in the biological fluid. The second substrate is stacked with the first substrate and includes a plurality of detectors vertically corresponding in position to the sampling apertures. Each of the detectors is configured to detect whether one of the macromolecules is present in a corresponding one of the sampling apertures and produce a detection output signal. A method for detecting macromolecules in a biological fluid is also disclosed. The apparatus has high integration and simple manufacturability, while the method is easy to use.