The chemical synapse transmits information from a neuron to another neuron in the neuronal network in the brain. The efficacy of synaptic transmission changes by modifying the number or size of synapses dynamically in cognitive functions. Thus, morphological analyses of synapses are of particular importance in neuroscience research. In the current study, we applied super-resolved three-dimensional stimulated emission depletion (3D-STED) microscopy for the morphological analyses of synapses. This approach allowed us to estimate the precise number of excitatory and inhibitory synapses in the mouse hippocampal tissue. Using this method, we discovered a region-specific increase in excitatory synapses in a model mouse of autism spectrum disorder, Neuroligin-3 KO. We detected an increase in excitatory synapses at the stratum oriens of hippocampal area CA1, although such an increase was not detected by conventional confocal microscopy. Our approach to estimating the synapse number will open a new field in developmental neuroscience.