Continuous cropping (CC) obstacles resulting from the increase in planting frequency restrict the sustainable development of the vegetable industry. To understand the mechanisms causing continuous cropping obstacles in-depth, we analyzed pepper (Capsicum annuum L.) rhizosphere soils. Samples were collected using a new type of in situ sampler composed of microporous ceramic tubes. After elution, the samples were qualitatively and quantitatively analyzed via gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). Then, the effects of the main possible allelochemicals on sensitive plants were investigated. Fourteen chemicals, including 2,4-di-tert-butyl-phenol (2,4-DTBP), diisobutyl adipate (DIBA), dibutyl phthalate (DBP) ，and limonene, were detected with GC-MS. The measured concentration of 2,4-DTBP increased with pepper growth. The results based on seed toxicity revealed that low-concentration (<50 mg·L-1) 2,4-DTBP improved lettuce seed germination rates and that high-concentration (>300 mg·L-1) 2,4-DTBP had significant (p<0.05) inhibitory effects. The results obtained from sensitive cucumber plants showed that the average plant height in the treatments containing 2,4-DTBP was lower (p<0.05). Significant increases in reactive oxygen species (ROS) levels and reductions in the photosynthetic rate (Pnet) were also detected (p<0.05). Leaf transpiration (Tr), stomatal conductance (Gs) and root activity also decreased significantly compared with those of the control. The comparison results between the 2,4-DTBP treatment and the pepper CC treatment showed that the cucumber was more significantly damaged in the latter. Similar trends also occurred in pepper. Our results indicate that the in situ sampler in this study can reflect the actual concentrations of possible allelochemicals in rhizosphere soil. 2,4-DTBP is one of the most important allelochemicals in pepper, and the accumulation of 2,4-DTBP may be an important factor inducing continuous cropping obstacles in pepper.