Detection of carbon dioxide (CO2) is of fundamental importance in diverse applications ranging from environmental analysis to agricultural production. In this work, a hybrid probe based on guanidinium-pendent oligofluorene (G-OF) and water-soluble conjugated polythiophene (PTP) has been developed for the turn on detection of CO2 with low background signal, taking advantage of the efficient fluorescence quenching of the tight aggregate of G-OF/PTP. In the presence of CO2, the electrostatic repulsion between G-OF and PTP can be effectively enhanced through protonation of the side chains, leading to the disaggregation and thus the “turn-on” fluorescence. The strategy allows for the light-up visible detection of CO2 with high sensitivity. Importantly, this system is capable of sensitively monitoring the concentration changes of CO2 in the process of the photosynthesis, which represents a concept to monitor the photosynthesis based on water-soluble conjugated polymers.