This study aimed to produce livestock manure biochar to decrease environmental problems from livestock manure and evaluate its effectiveness as an organic fertilizer by examining the growth and nutrient use efficiency of crops. A plot experiment was conducted to investigate the characteristics of lettuce growth and nitrogen use efficiency in upland soils treated with cow manure biochar. The cow manure biochar was applied at rates of 0, 3, 5, 7, and 10 t ha−1 (referred to as CMB0, CMB3, CMB5, CMB7, and CMB10, respectively), along with inorganic fertilizer (IF, NPK—200-59-12 kg ha−1). The lettuce cultivation test was carried out for 42 days, during which the fresh weight, dry weight, length, and number of lettuce leaves were measured. Nitrogen use efficiency was evaluated by determining the agronomic efficiency of N and the apparent recovery fraction of N. Overall, as the cow manure biochar application rate increased, crop growth and nitrogen uptake improved. Soils treated with CMB5 and CMB7 showed higher lettuce growth, nitrogen content, and nitrogen uptake compared to soils under other treatments. Nitrogen use efficiency followed a pattern similar to that of crop productivity, with cow manure biochar application levels playing a significant role. In particular, the agronomic efficiency of N and the apparent recovery fraction of N, which are both related to crop nutrient utilization, were significantly higher in the CMB5 treatment compared to the IF treatment. These results indicate that nitrogen use efficiency can be enhanced through biochar application when growing crops on agricultural land. Therefore, it is suggested that the appropriate application of cow manure biochar can reduce inorganic fertilizer use and increase crop productivity, thereby enabling sustainable and eco-friendly agriculture.