The soluble form of penicillin-binding protein 3 (sPBP 3∗) from Streptococcus pneumoniae was expressed in Escherichia coli as a six-histidine fusion protein. The protein was purified and utilized to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidaselabelled ampicillin (HRP-Amp) to the sPBP3∗ by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins), could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by European Union. Analysis of spiked milk samples showed acceptable recoveries ranged from 74.06% to106.31% in skimmed milk and 63.97-107.26% in whole milk, with coefficient of variations (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on the sPBP3∗ exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk.