The invention discloses screening and application of burkholderia gladioli real-time quantitative polymerase chain reaction (qRT-PCR) reference genes and primers thereof. According to obtained transcriptome analysis results and reported reference genes, 12 candidate reference genes with relatively stable expression are preliminarily screened, and gene names are shown as follows: atpD, clpP, clpX,ftsZ, gyrB, lpxC, pyrG, recA, rpoB , rpoD, thyA and 16S; for the candidate genes, amplification primers are designed; reference genes under different culture conditions (temperature, initial pH valueof medium, culture time, treatment with NaCl different in concentration) of burkholderia gladioli are screened; geNorm, NormFinder and Bestkeeper software is employed to analyze qRT-PCR results; and finally, the reference genes with most stable expression under different culture conditions and NaCl treatment condition are obtained by screening. The invention is beneficial to the stability and reliability of analysis and study of gene expression of the burkholderia gladioli under different conditions.