To optimize and determine the protein microarray detection condition for bovine lactoferrin (Lf) in milk.Methods Protein microarray technology and sandwich method was used to optimize the detection condition by probe experiment, homogeneity of the spots of the probes experiment, chessboard titration experiment, concentration dilution experiment, limit of detection and biologic limit of detection experiment. Results The 75# mouse monoclonal antibody was chosen as the printing probe, the pre-printing number was 46 points and the printing stable zone was from 46th to 90th point; the concentration of probe is 0.5 mg/ml, the valence of detection antibody was 1∶2 000; the dose-response relationship appeared the S curve from 0.6 to 612 ng/ml and the linearity range was from 9.56 to 306 ng/ml, respectively; the limit of detection and biologic limit of detection was 1.68 and 3.59 ng/ml, respectively; finally, the regression equation with an optimum determination coefficient (r=0.998) and the standard curve were established.Conclusion This study optimized the protein microarray detection condition for bovine Lf in milk, and these detection conditions were the basis of the further study for establishing the relevant protein microarray plat.