Lactoferrin (LF) has been proposed as a multifaceted functional ingredient of food. A competitive enzyme-linked immunosorbent assay (ELISA) was established to determine the LF in milk by using bovine LF antiserum and bovine LF-Biotin conjugate. The LF concentration in cow milk samples could be determined by this ELISA. However, the LF concentration in goat milk could not be measured using this method. Thus, the goat LF ELISA was established using goat LF-Biotin conjugate to measure the LF concentration in goat milk. Milk samples were collected for measurement of their LF concentrations. The mean LF level for the individual cow milk samples with the somatic cell count (SCC) below 1 × 105, 1 to 2.5 × 105 or 2.5 × 105 to 5 × 105 cells/mL was found to be about 176, 466 or 742 μg/mL, respectively. On the other hand, the LF level was about 200 μg/mL in goat bulk milks with the methylene blue reduction test (MBRT) time more than 5 hr. In conclusion, both LF ELISA methods developed in this study could be successfully used to determine the LF concentration in goat and cow milk.