A facile in-tube solid phase microextraction (in-tube SPME) procedure was developed to enrich ractopamine before HPLC-UV analysis. This was achieved by employing amide groups modified polysaccharide-silica hybrid monolith as an efficient sorbent. The monolith was synthesized by a simple reaction with agarose oxide and tetramethoxylisane, followed by the modification of amide groups via subsequent ring opening, “thiol–ene” click and dehydration reactions. Under the optimized extraction conditions, the enrichment factors for ractopamine, dopamine, clenbuterol, para-methylphenol and phenol were determined to be 50.5, 32.2, 4.8, 2.1 and 1.8, respectively. The monolithic column has ideal selectivity for ractopamine. Coupled with HPLC-UV, this method demonstrated a linearity within 2.0–800 ng/g for ractopamine with spiking in pork muscles (R2 = 0.9958). The LOD was 0.64 ng/g (S/N = 3) and recoveries ranged from 85.2 to 108.1% (n = 3). This approach provides a feasible way for analysis of trace ractopamine in biological samples.