Differences in variables of physical fitness and stroke performance by competition level (i.e., elite vs. sub-elite players) have not been systematically investigated yet. Thus, the objective of the systematic review with meta-analysis was to characterize and quantify competition-level dependent differences in physical fitness and stroke performance in healthy tennis players. A systematic literature search was conducted in the databases PubMed, Web of Science, and SportDiscus from their inception date till May 2022. Studies were included if they investigated healthy tennis players and reported at least one measure of physical fitness (e.g., lower extremity muscle power, endurance, agility, speed) or stroke performance (e.g., stroke velocity). Weighted standardized mean differences (SMD) were calculated and reported according to their magnitude. The search identified a total of N = 12,714 records, 16 of which met the inclusion criteria. Competition-level dependent differences in physical fitness and stroke performance were investigated by 11 and 10 studies, respectively. For physical fitness, moderate (lower extremity muscle power: SMD = 0.53; endurance: SMD = 0.59; agility: SMD = 0.54) and small (speed: SMD = 0.35) effects were detected; all in favour of elite tennis players. However, sub-group analyses revealed an influence of players' age showing higher SMD-values for adult than for young players. Further, a large effect (SMD = 1.00) was observed for stroke performance again in favour of elite tennis players. Lastly, a larger but not significantly different association between physical fitness and stroke performance was observed for elite (r = 0.562) compared to sub-elite (r = 0.372) tennis players. This systematic review and meta-analysis revealed better physical fitness and stroke performances in healthy elite compared to sub-elite tennis players. The greatest differences by competition level were shown in measures of lower extremity muscle power, endurance, and agility. Thus, training programs for sub-elite tennis players should place a special focus on these physical components.