Lactoferrin (LF), a bioactive multifunctional protein of the transferrin family, is found mainly in the secretions of all mammals, especially in milk. In the present study, a hybridoma cell (LF8) secreting IgG against bovine LF was screened, and the purified LF8 mAb showed high specificity and affinity to bovine LF. The linear range of ic-ELISA to detect LF was 9.76 ~ 625 ng/mL, with a limit of detection (LOD) of 0.01 ng/mL. The average recovery of intra- and inter-assay were (104.45 ± 4.12)% and (107.13 ± 4.72)%, respectively. The LOD of colloidal gold- and AuNFs-based strip by naked eye were 9.7 and 2.4 ng/mL, respectively, and the detection time was less than 10 min without any samples pretreatment and expensive equipment. The developed ELISA and lateral flow immunosensors based on specific IgG could be used directly for rapid detection of the bovine LF content in cow milk samples.