Owing to the biological significance of Cl in cells, several chemical fluorescent probes and biosensors have been constructed to monitor this anion in the cytosol and subcellular organelles. However, a fluorescent probe for the selective detection of nuclear Cl has not been described thus far. In the current study, we developed the first nuclear Cl-selective biosensor, Cl-YFP-NLS, whose fluorescence was effectively quenched by this anion, and demonstrated that it is an efficient and powerful tool for determining the levels of nuclear Cl. The results of cell studies using Cl-YFP-NLS as the probe suggested that the level of Cl in the nucleus is lower than that in the cytosol. In addition, Cl-YFP-NLS along with lysosomal (Lyso-MQAE) and mitochondrial Cl-selective fluorescent probes (Mito-MQAE) were utilized to determine the effects of various substances on the levels of Cl in subcellular organelles. The results showed that lysosomotropic agents decrease the lysosomal Cl concentration and increase the levels of mitochondrial and nuclear Cl. Also, observations suggested that substances capable of inducing mitochondrial outer membrane permeabilization without inducing lysosomal membrane permeabilization increase mitochondrial and nuclear Cl concentrations but they do not affect the level of lysosomal Cl. Moreover, a substance directly disrupting nuclear pore complexes increased the level of nuclear Cl and did not change the levels of lysosomal and mitochondrial Cl. Finally, nucleus-affecting substances that cause deoxyribonucleic acid damage and activate p53 and Bax increased the levels of mitochondrial and nuclear Cl without influencing the level of lysosomal Cl.