Aims: To remove humic substances from RNA extracted from soil for the study of bacterial gene expression in soil. Methods and Results: A soil RNA extraction method was improved by optimization of lysis conditions and further purification by a spin column, to efficiently remove humic substances that may hinder enzymatic reactions of extracted RNA. Fluorescence spectrophotometry demonstrated that the improved method removed both humic and fulvic acids efficiently. Using the improved method, the signal of gene expression detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) increased 10-fold compared with that using the previous method. Using the method, we extracted RNA from a sterilized field soil, which was inoculated with Pseudomonas putida KT2440 transformed with a chloroaromatic degrading plasmid, in the presence or absence of 3-chlorobenzoate (3CB). Real-time RT-PCR performed using the extracted RNA as a template confirmed the induction of chloroaromatic degrading genes in 3CB-amended soil. Conclusions: The modified soil RNA extraction method succeeded in removing the co-extracted humic substances from soil RNA efficiently and improving the detection efficiency of the bacterial gene expression in soil. Significance and Impact of the Study: This improved method is a useful tool for the extraction of RNA to detect gene expression in soil.