The invention belongs to the technical field of medicines and relates to engineering bacteria for mainly generating gentamicin C1a and a constructing method of the engineering bacteria. 6'-C-O-methyltransferase gntK in micromonospora echinospora of gentamicin generated bacteria is damaged by using a gene blocking technology, so that the synthesis of gentamicin C1, C2 and C2a is blocked and gentamicin C1a and small part of gentamicin C2b are mainly generated. The contents of the C1A, the C2, the C2a and C1 in an original strain are 6.5 percent, 45.3 percent, 20.1 percent and 27.1 percent respectively; and the contents of the C1a and the C2b in a blocked strain are 94.0 percent and 6.0 percent respectively. According to the constructing method, the gntK gene in the micromonospora echinospora is damaged by a method of deleting in the architecture to inactivate, including the construction of gntK gene blocking plasmid, conversion of the blocking plasmid into the micromonospora echinospora, screening of a double-exchange strain and analysis of a fermentation product. The engineering bacteria constructed by the constructing method provided by the invention have no resistant marker; and through continuous passage, the yield of the gentamicin C1a is very stable.