The invention discloses an Escherichia coli for producing riboflavin and constructing method and use of Escherichia coli. The preparation method comprises the following steps: designing ribosome bind sites RBS1, RBS2, RBS3, RBS4 and RBS5, respectively connecting the ribosome bind sites with genes ribA, ribB, ribD, ribE and ribC into fragments, splicing the fragments in order and enzyme-cutting and linking to a plasmid pTrc99a to obtain plasmid p20C-EC10, transferring into the Escherichia coli to obtain RF01S strain, transforming related gene of glycolytic pathway and ED pathway of the RF01S strain so as to improve the output of the riboflavin, and transforming the consumption pathway of the riboflavin to decrease the metabolism velocity of the riboflavin so as to further improve the riboflavin accumulation, inserting a Ptrc strong promoter into genome of the strain to obtain the Escherichia coli for producing the riboflavin. The constructed Escherichia coli strain has clear genetic background, and the glucose can be used as substrate to produce the riboflavin, and the shake-flask fermenting result is more than 1g/L, and a foundation is provided for improving the riboflavin output and yield.