[Objective] Generally, traditional λ-Red recombination system possessed low efficiency, complicated processes, inconsistent protocols, high false-positive rate and instability for multi-gene-knock-out/knock-in during manipulation on chromosome gene of Escherichia coli. In order to solve these problems, this study established a high efficiency and standard strategy of gene knock-out/in. [Methods] Based on λ-Red recombination system, new template plasmids were developed. A pSC101 derivative replication origin was used to diminish the false-positive problem. Convenient genetic manipulation was achieved by using high-copy-number plasmid and multiple cloning sites. New genetic marker was used to facilitate continuous multi-gene knock-out/in. A series of key targets within primary metabolite networks of E. coli were then knocked out/in using our methods. [Results] New λ-Red plasmids system, named SC101-Cre-LoxP-MCS system, was developed. The positive colonies were selected on two-resistance plate and 100% positive rate was achieved. [Conclusion] The efficiency of gene recombination was improved by the new method of gene knock-out/knock-in. This new system provides a rapid genetic manipulation. Our new strategy provides important insights into gene function research and genetic engineering bacteria with new genetic characteristics.