Escherichia coli bacteria widely recognize as a major treatment in water quality as diarrhea disease becoming one of leading causes of mortality worldwide. Carbon dots (CDs) modification study for detection of E. coli with the variation of colistin sulphate concentration has been conducted. The purpose of this research is to evaluate the effect of colistin sulphate concentration variation on fluorescence intensity for detecting E. coli bacteria in water samples. This study started by synthesizing CDs and CDs colistin variation concentrations of 1.0; 1.5; 2.0 and 2.5 mmol through carbonization of ammonium citrate and the addition of colistin. All precursors are characterized by using FT-IR. Morphological forms were evaluated with scanning emission microscopy (SEM) and transmission electron microscopy (TEM). CDs and CDs colistin solutions were measured their spectras with a UV-Vis spectrophotometer. Each of these CDs colistin was determined at the excitation wavelength ranges of 310-450 nm using a spectrofluorophotometer. Subsequently, CDs colistin was used for bacterial detection in samples with variation in E. coli bacteria concentration, thus analyzed by spectrofluorophotometer with an optimum excitation wavelength. The results showed that the highest intensity of fluorescence on CDs colistin occurred at the wavelength excitation of 360 nm. CDs colistin with the variation of colistin concentration 2.5 mmol revealed the highest fluorescence intensity. For the detection of E. coli, all CDs colistin produced the same trends of fluorescence spectra as the higher concentration of E. coli in the samples produced the higher intensity of emission.