The goal of this study was to develop an objective method for evaluation of ovarian follicle wall blood flow in cattle. Two subjective methods were used: (I) real-time ultrasound evaluations performed by one operator in the barn and (II) video clip evaluations performed by four operators in the laboratory. The following objective methods evaluated in the laboratory were used for comparison: (I) percentage of follicle wall circumference under blood flow (WUF) and (II) pixel area of color-Doppler signals. Cows (n = 21) were submitted to a synchronization protocol, follicles ≥7 mm were measured, and blood flow was evaluated every 12 h until ovulation using color-Doppler ultrasonography. No difference (P > 0.05) was observed among laboratory operators from day 2 of training onwards. Therefore, an average score of all operators was used for comparisons among different methods. Both subjective and objective methods of evaluation showed an increase (P < 0.0001) in follicle blood flow over time. Higher (P < 0.001) correlations were obtained between WUF and subjective laboratory evaluation than between WUF and pixel area or WUF and subjective barn data. Higher (P < 0.0003) correlation coefficients were observed for WUF than for the pixel area when compared with the barn (r = 0.70 vs. r = 0.42) or laboratory (r = 0.84 vs. r = 0.62) data. Subjective evaluations at the laboratory and barn produced stronger correlations with WUF (P < 0.0008) than with pixel area (P < 0.01). In conclusion, WUF is an effective and reliable method for objective evaluation of follicle wall blood flow in cows.