By using well-designed magnetic nanocomposites (MNCs) as probes, immunochromatographic test strip (ICTS) has shown some advantages in sensitivity and quantification of in vitro detection methods. In this study, MNCs were modified with controllable surface carboxyl groups through a facile two-step approach. 3-aminopropyltrimethoxysilane (APTS) was firstly grafted onto MNCs by a sol-gel reaction, followed by amide reaction between amino group of APTS and succinic anhydride. Density of controllable groups and conformation on the surface of MNCs were tuned by adjusting the APTS-to-MNCs ratio. Vibrio parahaemolyticus (VP) was used to examine the effect of surface modification of MNCs on the antibody conjugation and detection performance in ICTS. We also proposed the underlying mechanism based on the AFM analyses of antibody conjugation on two typical functionalized MNCs. It was found that with the increasing of APTS input, lower APTS-to-MNCs ratios (0.03 and 0.3μL/mg) can improve surface carboxylic groups’ density of MNCs. The optimal APTS-to-MNCs ratio (3μL/mg) promoted 1.4-fold antibody conjugating efficiency, and 3.4-fold signal-to-noise value in the ICTS for 2×105 CFU/mL VP as compared with the ratio of 0.03μL/mg. Whereas, due to the steric hindrance, the antibody conjugating efficiency and signal-to-noise value at high APTS-to-MNCs ratio (12μL/mg) respectively decreased 13.9% and 74.7% than the optimal ratio. Accordingly, the controlled surface functionalization of MNCs is the key for highly efficient and sensitive ICTS.