Abstract: Phospholipid fatty acids (PLFAs) were widely used as the biological mark to analysis the microbial community diversity qualitatively and quantitatively. Based on the Sherlock MIS(MIDI Inc.), A Perl script program PLFAEco was developed to analysis the PLFAs of the soil samples. The function of it concluded: calculation of the Shannon-Wiener (H1), Brillouin (H2) and McIntosh (H3) diversity index, the abundance (S), Pielou evenness (J) and Simpson index (D); calculation of the Mean and SD; analysis of Cluster and PCA and output the statistics graphs. Output was formated into a HTML webpage included the data table and graph, and the files(CSV format) were provided for data exchange with orther software. It took short time to complete PLFAs data analysis and was the powerful tool for data process attaching to the MIDI system. This program had been used to analysis the flue-cured tobacco soil and the stroma cushion of non-pollution pigsty. User can download the program, manual and the example data from www.dagri.org.