Cysteamine-modified silver nanoparticle aggregates has been fabricated for pentachlorophenol (PCP) sensing by surface-enhanced Raman spectroscopy (SERS) using a portable Raman spectrometer. The cysteamine monolayers could preconcentrate PCP close to the substrate surface through the electrostatic interaction, which makes the SERS detection of PCP possible. Moreover, the Raman bands of cysteamine could be used as the internal spectral reference in the quantitative analysis. Qualitative detection of PCP was carried out by SERS without any sample pretreatment. Quantitative analysis of PCP was further realized based on the prepared substrate, as the log-log plot of normalized SERS intensity of PCP versus its concentrations exhibits a good linear relationship. The SERS signals collected on 20 randomly selected points show that the relative standard deviation of the normalized Raman intensity is 5.8%, which indicates the substrate had good uniformity. The PCP sensor also shows good long-term stability in the analyte solution. The substrate was cyclic immersed into PCP and methanol solution; after several cycles, the sensor still had good adsorption to PCP, which revealed the sensor has good reusability. Coupling with a portable Raman spectrometer, the cysteamine-modified silver nanoparticle aggregates have the potential to be used for in situ and routine SERS analysis of PCP in environmental samples.