Biochar can improve nutrient retention and influence metal availability in soils. This study assessed the combined effects of Zn source, dose, and biochar amendment on lettuce growth and Zn uptake in a Zn-deficient acidic soil. Treatments included ZnSO₄ and a chelated Zn-glycine-NH₃ complex applied at agronomic (2 mg·kg⁻¹) and high (20 mg·kg⁻¹) Zn doses. Each treatment was tested with and without 5% biochar prepared by pyrolysis at 500 ºC of olive pruning waste. Biochar consistently enhanced lettuce fresh weight, particularly under high-dose Zn-glycine-NH₃ treatment, reaching increases of up to 79% compared to the corresponding soil-only treatment. High Zn fertilization did not negatively affect biomass, indicating tolerance of lettuce to elevated Zn inputs. In soil-only treatments, leaf Zn concentrations significantly increased at agronomic Zn doses—up to 293% above the control—whereas high Zn-glycine-NH₃ doses did not further enhance uptake. Conversely, in biochar-amended soils, Zn accumulation decreased markedly, with up to an 80% reduction under high chelate doses. These findings suggest that biochar improves plant growth by enhancing soil physical and chemical properties but may adsorb or immobilize Zn at high concentrations, reducing its bioavailability. Overall, the interaction between Zn source, application rate, and biochar strongly influences nutrient dynamics. Properly managed biochar application can improve crop performance while mitigating excessive Zn accumulation and potential environmental risks. This research was funded by the Ministerio de Ciencia, Innovación y Universidades, Agencia Es-tatal de Investigación y Fondo Europeo de Desarrollo Regional (Proyect PID2023-149789OB-I00 funded by MCIU /AEI /10.13039/501100011033 / FEDER, UE).