The chlorophyll content is an important experimental parameter in agronomy and plant biology research. In this report, we explore the feasibility of determining total concentration of extracts containing chlorophyll a and chlorophyll b by chlorophyll fluorescence. We found that an excitation at 457 nm results in the same integrated fluorescence emission for a molecule of chlorophyll a and a molecule of chlorophyll b. The fluorescence yield induced by 457 nm is therefore proportional to total molar chlorophyll concentration. Based on this observation, we designed an instrument to determine total chlorophyll concentrations. A single light emitting diode (LED) is used to excite chlorophyll extracts. After passing through a long-pass filter, the fluorescence emission is assessed by a photodiode. We demonstrate that this instrument facilitates the determination of total chlorophyll concentrations. We further extended the functionality of the instrument by including LEDs emitting at 435 and 470 nm wavelengths, thereby preferentially exciting chlorophyll a and chlorophyll b. This instrument can be used to determine chlorophyll a and chlorophyll b concentrations in a variety of organisms containing different ratios of chlorophylls. Monte-Carlo simulations are in agreement with experimental data such that a precise determination of chlorophyll concentrations in carotenoid-containing biological samples containing a concentration of less than 5 nmol/mL total chlorophyll can be achieved.