Background Synaptic degeneration is a central pathogenic event in Alzheimer’s disease that occurs early during the course of disease and correlates with cognitive symptoms. The pre-synaptic vesicle protein synaptotagmin-1 appears to be essential for the maintenance of an intact synaptic transmission and cognitive function. Synaptotagmin-1 in cerebrospinal fluid is a candidate Alzheimer biomarker for synaptic dysfunction that also may correlate with cognitive decline. Methods In this study, a novel mass spectrometry-based assay for measurement of cerebrospinal fluid synaptotagmin-1 was developed, and was evaluated in two independent sample sets of patients and controls. Sample set I included cerebrospinal fluid samples from patients with dementia due to Alzheimer’s disease ( N = 17, age 52–86 years), patients with mild cognitive impairment due to Alzheimer’s disease ( N = 5, age 62–88 years), and controls ( N = 17, age 41–82 years). Sample set II included cerebrospinal fluid samples from patients with dementia due to Alzheimer’s disease ( N = 24, age 52–84 years), patients with mild cognitive impairment due to Alzheimer’s disease ( N = 18, age 58–83 years), and controls ( N = 36, age 43–80 years). Results The reproducibility of the novel method showed coefficients of variation of the measured synaptotagmin-1 peptide 215–223 (VPYSELGGK) and peptide 238–245 (HDIIGEFK) of 14 % or below. In both investigated sample sets, the CSF levels of synaptotagmin-1 were significantly increased in patients with dementia due to Alzheimer’s disease ( P ≤ 0.0001) and in patients with mild cognitive impairment due to Alzheimer’s disease ( P < 0.001). In addition, in sample set I the synaptotagmin-1 level was significantly higher in patients with mild cognitive impairment due to Alzheimer’s disease compared with patients with dementia due to Alzheimer’s disease ( P ≤ 0.05). Conclusions Cerebrospinal fluid synaptotagmin-1 is a promising biomarker to monitor synaptic dysfunction and degeneration in Alzheimer’s disease that may be useful for clinical diagnosis, to monitor effect on synaptic integrity by novel drug candidates, and to explore pathophysiology directly in patients with Alzheimer’s disease.