<b><i>Introduction:</i></b> Biomarkers of Alzheimer’s disease (AD) that can easily be measured in routine health checkups are desirable. Urine is a source of biomarkers that can be collected easily and noninvasively. We previously reported on the comprehensive profile of the urinary proteome of AD patients and identified proteins estimated to be significantly increased or decreased in AD patients by a label-free quantification method. The present study aimed to validate urinary levels of proteins that significantly differed between AD and control samples from our proteomics study (i.e., apolipoprotein C3 [ApoC3], insulin-like growth factor-binding protein 3 [Igfbp3], and apolipoprotein D [ApoD]). <b><i>Methods:</i></b> Enzyme-linked immunosorbent assays (ELISAs) were performed using urine samples from the same patient and control groups analyzed in the previous proteomics study (18 AD and 18 controls, set 1) and urine samples from an independent group of AD patients and controls (13 AD, 5 mild cognitive impairment [MCI], and 32 controls) from the National Center for Geriatrics and Gerontology Biobank (set 2). <b><i>Results:</i></b> In set 1, the crude urinary levels of ApoD, Igfbp3, and creatinine-adjusted ApoD were significantly higher in the AD group relative to the control group (<i>p</i> = 0.003, <i>p</i> = 0.002, and <i>p</i> = 0.019, respectively), consistent with our previous proteomics results. In set 2, however, the crude urinary levels of Igfbp3 were significantly lower in the AD+MCI group than in the control group (<i>p</i> = 0.028), and the levels of ApoD and ApoC3 did not differ significantly compared to the control group. Combined analysis of all samples revealed creatinine-adjusted ApoC3 levels to be significantly higher in the AD+MCI group (<i>p</i> = 0.015) and the AD-only group (<i>p</i> = 0.011) relative to the control group. <b><i>Conclusion:</i></b> ApoC3 may be a potential biomarker for AD, as validated by ELISA. Further analysis of ApoC3 as a urinary biomarker for AD is warranted.