Background Carnitine-acylcarnitine translocase (CACT) deficiency is a rare autosomal recessive metabolic disorder of mitochondrial long-chain fatty acid oxidation. Newborn screening via tandem mass spectrometry (MS/MS) technology enables early diagnosis. However, previous analyses of MS/MS data of patients showed that some results were misdiagnosed because they did not show typical acylcarnitine profiles of CACT deficiency. This study aimed to identify additional indices to assist the diagnosis of CACT deficiency. Methods To evaluate the acylcarnitine profile and the acylcarnitine ratios of individuals with CACT deficiency, the MS/MS data of 15 patients diagnosed via genetic testing were retrospectively analysed. The sensitivity and false-positive rates of primary acylcarnitine markers and ratio indices were validated using the data from 28,261 newborns and 53 false-positive cases. Additionally, the MS/MS data of 20 newborns carrying the c.199-10T>G mutation in SLC25A20 and 40 normal controls were compared to verify whether the carriers had abnormal acylcarnitine concentrations. Results The acylcarnitine profiles from 15 patients were classified into three categories using C12, C14, C16, C18, C16:1, C18:1, and C18:2 as the primary diagnostic markers. The first category represented a typical profile (P1–P6). The second category for patients P7 and P8 showed a significant decrease in the C0 level and a normal concentration of long-chain acylcarnitines. The third category for patients P9–P15 showed the presence of interfering acylcarnitines. The second and third categories may have been misdiagnosed. An acylcarnitine ratio analysis showed that C14/C3, C16/C2, C16/C3, C18/C3, C16:1/C3, and C16:1-OH/C3 were significantly increased in all 15 patients. The verification of 28,261 newborn screening results showed that the false-positive rate of ratios, except for (C16 + C18)/C0, was lower than that of acylcarnitine indices (0.02–0.08% vs. 0.16–0.88%). None of the single long-chain acylcarnitines could separate patients from the false-positive cases; however, all ratios produced good discrimination between the two groups. Conclusions Based on the primary acylcarnitine markers alone, CACT deficiency can be misdiagnosed in newborn screening. The ratios of the primary markers (C16 + C18:1)/C2, C16/C2, C16:1/C3, and C16:1-OH/C3 can facilitate the diagnosis of CACT deficiency, thereby increasing sensitivity and reducing false-positivity.