Abstract The anaerobic co-digestion (AcoD) of organic substrates has reportedly shown synergistic effects, but the current indices used to evaluate the magnitude of such effects cannot well support the findings and in some cases bring about confusion. In this paper, meta-analysis was performed through a database compiled from 124 peer-reviewed articles focused on the AcoD of livestock manure. Six commonly used indices for evaluating synergistic effects were statistically analyzed in order to obtain a representative evaluation index. Moreover, the synergy intervals of substrate mixing were preliminarily analyzed on element and molecular levels using the optimized index. The results indicated that the ratio of experimental values to weighted values is a relatively optimized evaluation index. Methane production and organic degradation were found to be two independent indices, thus both of them should be considered simultaneously when AcoD process is under consideration. From a statistical standpoint, the recommended synergy interval of carbon-nitrogen ratio is 20–27. A higher probability of synergy can be achieved when the lipid/carbohydrate ratio is higher than 0.13, as well as the protein/carbohydrate ratio is greater than 0.26. Moreover, a relatively complete execution strategy of AcoD has been preliminarily proposed in terms of selection of co-substrate, mixing ratio, and synergy evaluation. The results achieved herein would have some biases and uncertainties due to the fact that only few studies explicitly demonstrated carbohydrate forms, especially lignin. Therefore, the evaluated indices should be further verified and improved by including more details about carbohydrate forms.