The in situ biological process relays on a high performance attenuated total reflection (ATR) probe while ATR technique is a powerful tool for inline analysis. The basic principle and characteristic of the ATR technique was analyzed in this paper. A low cost and high throughout ATR probe was designed which can be inserted into the biological reactor directly with nondestructive and zero delay monitoring. It shows that the online ATR probe has a wide spectrum range, and the collected spectra has a high signal-to-noise ratio, which can be used in in-line quantitative analysis for biological process.