The increasing emergence of multi-drug resistant poses a serious threat to public health and food safety issues worldwide. Bacteriophage lysins are promising alternatives to antibiotics. Here, we describe an efficient method for rapid screening of engineered chimeric lysins and report a unique “Antimicrobial peptide(AMP)” chimeric lysins, AP-LyS1, with robust activity and an extended-spectrum Staphylococcus host range against most Staphylococcus species (including MRSA and MRCNS). AP-LyS1 is the lysin that demonstrates activity against bovine mastitis-causing pathogens Staphylococcus intracellular of MAC-T cell line. This study demonstrates the success of the screening method resulting in a powerful lysin with potential for treating most Staphylococcus associated infections.