To assess the effect of tear film instability in dry eye disease (DED) by measuring visual performance and tear film optical quality in a simultaneous real-time analysis system., Thirty-seven DED participants and 20 normal controls were recruited. A simultaneous real-time analysis system was developed by adding a functional visual acuity (FVA) channel to a double-pass system. Repeated measurements of FVA and objective scatter index (OSI) were performed simultaneously with this system under blink suppression condition for 20 s. Patient-reported symptoms was evaluated using the Ocular Surface Disease Index (OSDI) questionnaire. Mean FVA, mean OSI, and visual acuity break-up time were defined. The OSI maintenance ratio was calculated as an evaluation index to assess the difference between dynamic OSI changes and baseline OSI. The visual maintenance ratio was also calculated in the same way., Moderate correlations were noted between mean OSI and FVA-related parameters (mean FVA, visual maintenance ratio, visual acuity break-up time: 0.53, - 0.56, - 0.53, respectively, P < 0.01 for all). Moderate to high correlations were noted between OSI maintenance ratio and FVA-related parameters (mean FVA, visual maintenance ratio, visual acuity break-up time: - 0.62, 0.71, 0.64, respectively, all P < 0.01). The metrics derived from the simultaneous real-time analysis system were moderately correlated with the patient-reported symptoms and the visual acuity break-up time possessed the highest correlation coefficients with OSDI total, ocular symptoms, and vision-related function (- 0.64, - 0.63, - 0.62, respectively, P < 0.01). The OSI-maintenance ratio alone appeared to exhibit the best performance of the metrics for the detection of DED with sensitivity of 95.0% and specificity of 83.8% and the combinations of FVA parameters and OSI parameters were valid and can further improve the discriminating abilities., OSI-related metrics were found to be potential indicators for assessing and diagnosing DED which correlated with both subjective visual performance and patient-reported symptoms; the FVA-related metrics were quantifiable indicators for evaluating visual acuity decline in DED., Chinese Clinical Trial Registry, ChiCTR2100051650. Registered 29 September 2021, https://www.chictr.org.cn/showproj.aspx?proj=134612.