The Escherichia coli (E. coli) has been widely employed in biotechnology industry and academia. However, the bioproduct manufacturing driven by E. coli is prone to the phage contamination. Good laboratory/factory hygiene may decrease but not avoid completely the chances of the phage contamination. The present study aims to resolve this problem by engineering laboratory/factory-specific phage-resistant E. coli strains. By adding a laboratory or factory derived phage into the atmospheric and room temperature plasma mutagenized E. coli, a phage-resistant strain could be generated. Interestingly, the resistant strain exhibited cross-resistance to unencountered phages. When operating the resistant strain in a polluted environment, the phage contamination was largely prevented. There was no significant difference in heterogeneous protein production between the parental strain and the phage-resistant strain. Importantly, it requires only one day to generate the phage-resistant strain. This practical method for engineering laboratory/factory-specific phage-resistant strains may have great potential in resuming E. coli operation in laboratories and factories during phage contamination outbreaks.