The application of organic fertilizer is one of the important measures to improve the quality of tea and increase the yield of tea. The utilization of cow manure resources can help to reduce the pollution of the agricultural ecological environment. In order to explore the impact of cow manure organic fertilizer on the yield and quality of tea, through two consecutive years of field trials (2019—2020), the effects of different ratios of cow manure organic fertilizer replacing chemical fertilizer on the internal and external quality and yield of Lu’an Guapian Wuniuzao tea and the soil fertility of tea gardens were studied, and the suitable ratio of cow manure organic fertilizer replacing chemical fertilizer for tea gardens was selected. The results showed that the yield of Wuniuzao tea was the highest in all chemical fertilizer treatments, at 821.87 kg/hm², followed by the treatment of organic fertilizer with cow manure completely replacing chemical fertilizers, at 817.13 kg/hm². With the increase in the replacement ratio of cow manure organic fertilizer, the yield of Wuniuzao tea showed an upward trend, and the incremental output of Wuniuzao tea was 20.40%~44.01% in the fertilization treatment; In treatments of the organic fertilizer of cow manure replaced 70% nitrogen fertilizer and the organic fertilizer of cow manure replaced all nitrogen fertilizers, the internal composition and sensory quality of Wuniuzao tea all scored the best; The replacement of chemical fertilizers with cow manure organic fertilizer could increase the pH of tea garden soil. With the increase of the replacement ratio of cow manure organic fertilizer, soil organic matter and total nitrogen content all showed an upward trend, and alkaline nitrogen, available potassium, and available phosphorus content all showed an increase first and then declining trend, and reached the highest under the treatment of cow manure organic fertilizer replacing 70% nitrogen fertilizer; Cow manure organic fertilizer replacing chemical fertilizer increased the activity of tea garden soil urease, invertase, acid phosphatase and protease, and the soil enzymes activities were the highest under treatment that cow manure organic fertilizer replaced 70% nitrogen fertilizer. Comprehensively considering the effects of replacing chemical fertilizers with cow manure organic fertilizers on the yield, quality, biological characteristics, and soil fertility of Wuniuzao tea, replacing 70%-100% nitrogen fertilizers with cow manure organic fertilizers is a better model for replacing chemical fertilizers with organic fertilizers in the tea garden system.