Instead of indexing to access the rows, you could use iteration:
l = sum(row[i] for i, row in enumerate(matrix))
Instead of doing two passes over matrix to calculate l and r, you could accumulate the difference in one pass: (note also negative indexing from end of list)
difference = sum(row[i] - row[-i-1] for i, row in enumerate(matrix))
return abs(difference)
The single-pass approach would also allow you to process input line by line, instead of storing the whole matrix in memory.