It is assumed that the concentration of amyloid-β1-40 (Aβ1-40) in cerebrospinal fluid (CSF) reflects the total amount of Aβ protein in the brain and thus allows a better interpretation of inter-individual differences in Aβ quantity than the Aβ1-42 concentration. In this study, Aβ1-40 was added to the existing CSF biomarker panel of Aβ1-42, total tau (T-tau), and phosphorylated tau (P-tau181P) in order to test whether the accuracy of the differential dementia diagnosis improved. The concentration of Aβ1-40 (INNOTEST® β-amyloid(1-40) prototype version, Innogenetics NV, Belgium) and the other biomarkers (INNOTEST®) was determined in CSF samples from 80 Alzheimer's disease (AD) patients, 75 non-AD dementia patients, and 30 controls. A large proportion of the study population had autopsy-confirmed neurodegeneration (AD: 73/80 = 91%; non-AD: 38/75 = 51%). The levels of Aβ1-40 were decreased in AD (10856 ± 4745 pg/mL) and non-AD patients (10519 ± 4491 pg/mL) when compared to controls (14760 ± 7846 pg/mL) (p = 0.002 and p = 0.001). The Aβ1-42/Aβ1-40 ratio was significantly decreased in AD (0.043 ± 0.021) as compared to non-AD patients (0.064 ± 0.027; p < 0.001) and controls (0.053 ± 0.023; p < 0.001). In order to differentiate AD from non-AD patients, a decision tree was constructed. The diagnostic accuracy of the decision tree that contained Aβ1-42, Aβ1-40, P-tau181P, and the Aβ1-42/Aβ1-40 ratio was significantly better than the diagnostic accuracy (80% versus 74%) of the decision tree without Aβ1-40 and the Aβ1-42/Aβ1-40 ratio (p < 0.001). In conclusion, no difference in Aβ1-40 CSF levels was found between AD and non-AD patients, but adding CSF Aβ1-40 and the CSF Aβ1-42/Aβ1-40 ratio to a biomarker-based decision tree, might have an added value for discriminating AD from non-AD patients in case of intermediate CSF P-tau181P values.