The retrieval from space of a very weak fluorescence signal was studied in the O2A and O2B oxygen atmospheric absorption bands. The accuracy of the method was tested for the retrieval of the chlorophyll fluorescence and reflectance terms contributing to the sensor signal. The radiance at the top of the atmosphere was simulated by means of a commercial radiative-transfer program at a high resolution (0.1 cm−1). A test data set was generated in order to simulate sun-induced chlorophyll fluorescence at the top of the canopy. Reflectance terms were spectrally modeled using cubic splines and fluorescence by means of the sum of two Voigt functions. Sensor radiance residual minimization was performed in the presence of a multiplicative noise, thus ensuring that the sensor simulations were realistic. The study, which focused on the possibility of retrieving fluorescence with an accuracy better than 10%, was performed for instrument resolutions ranging from about 0.4 cm−1 to 2 cm−1 in order to test the algorithm for the characteristics of existing and planned hyper-spectral sensors. The algorithm was also used to retrieve fluorescence in the single O2A band at the OCO and TANSO-FTS instrument spectral resolutions.