Objective To explore the effect of dilution method on the interpretation of multi-karyotypes in anti-nuclear antibody (ANA). Methods From June to August 2019, 56 nuclear composite karyotypes and 10 nuclear monokaryotypes from patients admitted to Nanjing Drum Tower Hospital were collected. Western blotting was used to detect anti-nuclear antibody (IgG) spectra, and indirect immunofluorescence method was used to detect anti-nuclear antibody immunoglobulin G (IgG) diluted at 1∶100, 1∶1000, and 1∶10 000. The maximum dilution titer of fluorescence was determined, and the differences of karyotype and titer interpretation results before and after dilution were compared combined with ANA spectrum results. Results There was no change in karyotype and titer of 10 nuclear mono-karyotype samples before and after dilution. Among 56 nuclear compound karyotypes, 8 cases (14.2%) were increased, 3 cases(5.4%) were decreased, and 45 cases (80.4%) remained unchanged. The titer analysis showed that 23 cases (41.1%)were increased, 8 cases (14.2%) were decreased, 2 cases (3.6%) were partially increased and partially decreased, and 23 cases (41.1%) had no change. This result was more correlated with ANA spectrum results. Conclusion The method of dilution can help to determine the karyotypes and titers of nuclear multi-karyotypes, so as to improve the accuracy of the results.