Background: Because of the low sensitivity of commercial products, development of a facile method to rapidly identify plague on-site remains highly attractive. Line arrays of poly(methacrylic acid) (PMAA) brushes were grafted using a photoresist template to fabricate one-dimensional diffraction gratings (DGs). The as-prepared samples first bound protein G to immobilize and orient the tails of the antibody of Yersinia pestis (abY). A laser beam was employed to analyze the 2D and 3D reflective signals of DGs at an incident angle of 45°. The abY-tailed PMAA DG possessed an optical feature with a characteristic diffraction effect along the SII, in which the projection of the laser beam on the plane of the DG chip was parallel to the strips, and ST configurations, in which they were perpendicular. A fluidic diffraction chip based on the abY-tailed PMMA DG was fabricated to examine the ability to detect Yersinia pestis along the ST configuration. Results: Upon flowing through the chip, Yersinia pestis was attached to the abY-tailed PMMA DG, which changed the diffraction intensity. The degree of the diffraction intensity exhibited a linear response to Yersinia pestis at concentrations from 102 to 107 CFU mL−1, and the limit of detection was 75 CFU mL−1, 1000 times lower than a commercial product (Alexter Bio-Detect Test). The diffractive sensor could selectively detect Yersinia pestis in spiked serum samples, with excellent standard deviation and recovery. Conclusion: Our platform provides a simple, label-free method for on-site plague diagnosis to prevent the highly rapid transmission of plague.