In order to study the spatial distribution of carbon (C) and nitrogen (N) acquiring hydrolase activity hotspots in rhizosphere in karst ecosystems, we incubated maize (one of main grain crops in locality), alfalfa (forage grass), and sedge (common grass in abandoned farmland) in rhizoboxes, which were filled with the collected topsoil of sloping farmland in a karst region of Guizhou province, China. The distribution of β-glucosidase (βG) and N-acetyl glucoaminosidase (NAG) activities in rhizosphere and non-rhizosphere soils was in situ detected by zymography. The results show that: 1) the hotspots of βG and NAG activities were located at both tip and elongation zones of roots for the three plants. The largest hotspot extent ranked in order: alfalfa > sedge > maize. 2) The hotspot extent of βG and NAG activities in maize rhizosphere was similar in size between root tip and elongation zones (about 1.13 mm). Alfalfa had larger hotspot extent of C-acquiring hydrolase activity in its root elongation zone (1.98 mm) than in root tip zone (1.19 mm), but larger hotspot extent of N-acquiring hydrolase activity in root tip zone (0.91 mm) than in root elongation zone (0.59 mm). Sedge had larger hotspot extent for both βG and NAG activities in root tip zone (1.38-1.86 mm) than root elongation zone (0.93-1.16 mm). 3) Rhizosphere microbes of leguminous alfalfa with N-fixing capacity preferred carbon, probably due to their low demand for nitrogen. Rhizosphere microbes of sedge preferred nitrogen, which might be caused by nitrogen limitation in the karst soil. On the whole, alfalfa had largest hotspot extent of βG and NAG activities activities in rhizosphere, where microbes preferred C to meet requirements for N fixation. Therefore, planting alfalfa can alleviate nitrogen limitation for ecological restoration in abandoned sloping farmland in karst regions. The in situ zymography in this study provides a quantitative tool for exploring microbial nutrient-acquiring mechanisms in rhizosphere.