Chromium is a controversial element with an important essentiality and toxicity. Depending on its different species, its speciation analysis in bio-origin matrices is of utmost importance. Ammonium pyrrolidine dithiocarbamate (APDC) and layered double hydroxide (LDH) nanoparticles, with the purpose of combining their outstanding performances, were served to speciate chromium ions in human biological samples. The as-obtained sorbent (nano LDH-APDC) - after characterization by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, thermal gravimetric analysis (TGA), and scanning electron microscopy (SEM) - was used as a novel pH-sensitive adsorbent in an integrated one-step dispersive solid-phase extraction (I-OS-DSPE), which combines the benefits of the air-assisted microextraction and dispersive solid-phase extraction methods. An interesting feature of the nano LDH-APDC sorbent is that it is dissolved in an aqueous solution when the pH of the solution is lower than 4. Thus the analyte elution step, as required in most of the sorbent-based extraction methods, was obviated by dissolving the sorbent in an acidic solution after extraction and separation from the sample solution. The Cr(VI) ions were first extracted, while the Cr(III) ions remained in the aqueous solution. The extract was then directly injected into a flame atomic absorption spectrometer with a micro-sampling introduction system, and the concentration of the Cr(III) ions was calculated by its subtraction from the total chromium ions present. Several variables including the pH (5), type and amount of the nanosorbent used (30 mg of nano (Zn-Al) LDH-APDC), number of extraction cycles (15 times), and elution conditions (200 µL of 6.0 mol L−1 HNO3) were investigated to achieve the maximum extraction efficiency. Under the optimum experimental conditions, the limit of detection, linear range, consumptive index, and enrichment factor for the Cr(VI) ions were 2.4 μg L−1, 8.0–640 μg L−1, 0.24, and 42.5 ± 1, respectively. These findings suggested that nano (Zn-Al) LDH-APDC could be regarded as a promising adsorbent for an efficient speciation of the chromium species in the human hair, nail, saliva, plasma, and urine samples.