Endogenous hormones play an important role in the growth and development of roots. The objective of this research was to study the effect of four types of N fertilizers on the root growth of strawberry (Fragaria ananassa Duchesne) and the endogenous enzymes of indole-3-acetic acid (IAA), abscisic acid (ABA), and isopentenyl adenosine (iPA) in its roots and leaves using enzyme-linked immunosorbent assay. Application of all types of N fertilizers significantly depressed (P ≤ 0.05) root growth at 20 d after transplanting. Application of organic-inorganic fertilizer (OIF) as basal fertilizer had a significant negative effect (P ≤ 0.05) on root growth. The application of OIF and urea lowered the lateral root frequency in strawberry plants at 60 d (P ≤ 0.05) compared with the application of two organic fertilizers (OFA and OFB) and the control (CK). With the fertilizer treatments, there were the same concentrations of IAA and ABA in both roots and leaves at the initial growth stage (20 d), lower levels of IAA and ABA at the later stage (60 d), and higher iPA levels at all seedling stages as compared to those of CK. Thus, changes in the concentrations of endogenous phytohormones in strawberry plants could be responsible for the morphological changes of roots due to fertilization.